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Objective: OA is a very heterogeneous disease. Here, we aimed to differentiate OA patients based on their serum-induced cell-integrated signalling patterns., Design: In order to monitor the activity of different cellular homeostasis-regulating pathways in response to patient serum, we analysed the response of human OA serum samples to sixteen cell-based transcription factor luciferase reporter assays. In this study we compared serum samples from 55 patients with knee OA, 56 patients with hand OA and 42 healthy controls., Results: Differential serum-induced pathway activity was observed between samples from healthy controls, knee OA and hand OA patients: Serum of hand OA patients induced high MAPK-related AP1 activity whereas serum of knee OA patients induced more SRE, ISRE and SOX9 activity, which is related to ELK1-SRF, STAT1-STAT2 and SOX9 activity respectively. Principal component analysis revealed that these differences differentiate hand OA from knee OA. Both hand and knee OA clustered clearly in 2 different endotypes each, but no principle component could be identified of these subtypes within either the hand OA or the knee OA sample group., Conclusion: This study demonstrates that serum from hand OA and knee OA patients evokes diverse cellular signalling patterns that differentiates hand OA, knee OA and healthy controls. This underlines that the pathomolecular mechanisms of OA are likely significantly different between hand and knee OA, a finding that could lead to new insight into the pathobiology of OA endotypes and joint-specific therapies., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: Patient-specific instrumentation (PSI) is a commonly used technique designed to improve mechanical alignment in total knee arthroplasty (TKA) and was therefore believed to lead to better clinical outcome and implant survival rates compared with conventional instruments (CIs). To date, long-term results comparing these two techniques are not available., Methods: This study is a 10-year follow-up of a previous double-blind multicenter randomized controlled trial where PSI was compared with CI. Patients with osteoarthritis of the knee who were candidates for TKA were included. Exclusion criteria were metal near the knee-, ankle- or hip joint, patients with contra-indications for a magnetic resonance imaging (MRI) scan and patients who had previous knee surgery (except arthroscopic meniscectomy). Clinical outcomes were assessed using patient-reported outcome measures (PROMs), and the analysis was performed with a general linear mixed model for repeated measurements. Kaplan-Meier curves were used to compare revision rates. X-rays were obtained and examined by two individual reviewers for any signs of loosening of the components., Results: At a mean follow-up of 10.1 (SD 0.1) years, 129 patients (loss to follow-up 23%) were analysed in this trial. No statistically significant difference between the two groups were found for any of the PROMs and revision rates were comparable, six in the PSI group and three in the CI group (p = 0.29). Two X-rays in the PSI group showed a radiolucent line of the femoral component., Conclusion: At 10-year follow-up, PSI does not lead to better clinical outcome or survival of the prosthesis compared with CI., (© 2024 European Society of Sports Traumatology, Knee Surgery and Arthroscopy. Published by John Wiley & Sons Ltd.)

Objective: We aimed to characterize calcium-containing crystals present in synovial fluid from patients with knee osteoarthritis (OA) using Raman spectroscopy, and specifically investigate the biological effects of calcite crystals., Design: Thirty-two synovial fluid samples were collected pre-operatively from knee OA patients undergoing total joint arthroplasty. An integrated Raman polarized light microscope was used for identification of crystals in synovial fluid. Human peripheral blood mononuclear cells (PBMC's), human OA articular chondrocytes (HACs) and fibroblast-like synoviocytes (FLSs) were exposed to calcite crystals. Expression of relevant cytokines and inflammatory genes were measured using enzyme-linked immuno sorbent assay (ELISA) and real-time polymerase chain reaction (PCR)., Results: Various calcium-containing crystals were identified, including calcium pyrophosphate (37.5 %) and basic calcium phosphate (21.8 %), but they were never found simultaneously in the same OA synovial fluid sample. For the first time, we discovered the presence of calcite crystals in 93.8 % of the samples, while dolomite was detected in 25 % of the cases. Characterization of the cellular response to calcite crystal exposure revealed increased production of innate immune-derived cytokines by PBMC's, when co-stimulated with lipopolysaccharide (LPS). Additionally, calcite crystal stimulation of HACs and FLSs resulted in enhanced secretion of pro-inflammatory molecules and alterations in the expression of extracellular matrix remodeling enzymes., Conclusions: This study highlights the unique role of Raman spectroscopy in OA crystal research and identified calcite as a novel pro-inflammatory crystal type in OA synovial fluid. Understanding the role of specific crystal species in the OA joint may open new avenues for pharmacological interventions and personalized approaches to treating OA., Competing Interests: Declaration of Competing Interest Cees Otto: Shareholder of Hybriscan Technologies B.V., a company which produces and sells Raman spectrometer devices, including H-iRPolM which was used for this study. Matthijs Janssen, Tim L Jansen: Shareholders of Crystalytics B.V., a company interested in development of tools used for clinical identification of synovial crystals. Tim Welting: Shareholder of Chondropeptix, Inventor of: WO2017/178251, WO2017/178253, WO2023/280615. Guus van den Akker: Inventor of WO2023/280615. All other authors have no potential conflicts of interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Raman spectroscopy is proposed as a next-generation method for the identification of monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals in synovial fluid. As the interpretation of Raman spectra requires specific expertise, the method is not directly applicable for clinicians. We developed an approach to demonstrate that the identification process can be automated with the use of machine learning techniques. The developed system is tested in a point-of-care-setting at our outpatient rheumatology department., Methods: We collected synovial fluid samples from 446 patients with various rheumatic diseases from three centra. We analyzed all samples with our Raman spectroscope and used 246 samples for training and 200 samples for validation. Trained observers classified every Raman spectrum as MSU, CPP or else. We designed two one-against-all classifiers, one for MSU and one for CPP. These classifiers consisted of a principal component analysis model followed by a support vector machine., Results: The accuracy for classification of CPP using the 2023 ACR/EULAR CPPD classification criteria was 96.0% (95% CI 92.3-98.3), while the accuracy for classification of MSU with using the 2015 ACR/EULAR gout classification criteria was 92.5% (95% CI 87.9-95.7). Overall, the accuracy for classification of pathological crystals was 88.0% (95% CI 82.7-92.2). The model was able to discriminate between pathologic crystals, artifacts, and other particles such as microplastics., Conclusion: We here demonstrate that potentially complex Raman spectra from clinical patient samples can be successfully classified by a machine learning approach, resulting in an objective diagnosis independent of the opinion of the medical examiner., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Currently available focal knee resurfacing implants (FKRIs) are fully or partially composed of metals, which show a large disparity in elastic modulus relative to bone and cartilage tissue. Although titanium is known for its excellent osseointegration, the application in FKRIs can lead to potential stress-shielding and metal implants can cause degeneration of the opposing articulating cartilage due to the high resulting contact stresses. Furthermore, metal implants do not allow for follow-up using magnetic resonance imaging (MRI).To overcome the drawbacks of using metal based FKRIs, a biomimetic and MRI compatible bi-layered non-resorbable thermoplastic polycarbonate-urethane (PCU)-based FKRI was developed. The objective of this preclinical study was to evaluate the mechanical properties, biocompatibility and osteoconduction of a novel Bionate® 75D - zirconium oxide (B75D-ZrO 2 ) composite material in vitro and the osseointegration of a B75D-ZrO 2 composite stem PCU implant in a caprine animal model. The tensile strength and elastic modulus of the B75D-ZrO 2 composite were characterized through in vitro mechanical tests under ambient and physiological conditions. In vitro biocompatibility and osteoconductivity were evaluated by exposing human mesenchymal stem cells to the B75D-ZrO 2 composite and culturing the cells under osteogenic conditions. Cell activity and mineralization were assessed and compared to Bionate® 75D (B75D) and titanium disks. The in vivo osseointegration of implants containing a B75D-ZrO 2 stem was compared to implants with a B75D stem and titanium stem in a caprine large animal model. After a follow-up of 6 months, bone histomorphometry was performed to assess the bone-to-implant contact area (BIC). Mechanical testing showed that the B75D-ZrO 2 composite material possesses an elastic modulus in the range of the elastic modulus reported for trabecular bone. The B75D-ZrO 2 composite material facilitated cell mediated mineralization to a comparable extent as titanium. A significantly higher bone-to-implant contact (BIC) score was observed in the B75D-ZrO 2 implants compared to the B75D implants. The BIC of B75D-ZrO 2 implants was not significantly different compared to titanium implants. A biocompatible B75D-ZrO 2 composite approximating the elastic modulus of trabecular bone was developed by compounding B75D with zirconium oxide. In vivo evaluation showed an significant increase of osseointegration for B75D-ZrO 2 composite stem implants compared to B75D polymer stem PCU implants. The osseointegration of B75D-ZrO 2 composite stem PCU implants was not significantly different in comparison to analogous titanium stem metal implants., (© 2024 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-β2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-β2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-β2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-β2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-β2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-β2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

Chronic lower back pain caused by intervertebral disc degeneration and osteoarthritis (OA) are highly prevalent chronic diseases. Although pain management and surgery can alleviate symptoms, no disease-modifying treatments are available. mRNA delivery could halt inflammation and degeneration and induce regeneration by overexpressing anti-inflammatory cytokines or growth factors involved in cartilage regeneration. Here, we investigated poly(amidoamine)-based polymeric nanoparticles to deliver mRNA to human joint and intervertebral disc cells. Human OA chondrocytes, human nucleus pulposus (NP) cells, human annulus fibrosus (AF) cells, fibroblast-like synoviocytes (FLS) and M1-like macrophages were cultured and transfected with uncoated or PGA-PEG-coated nanoparticles loaded with EGFP-encoding mRNA. Cell viability and transfection efficiency were analyzed for all cell types. Nanoparticle internalization was investigated in FLS and M1-like macrophages. No significant decrease in cell viability was observed in most conditions. Only macrophages showed a dose-dependent reduction of viability. Transfection with either nanoparticle version resulted in EGFP expression in NP cells, AF cells, OA chondrocytes and FLS. Macrophages showed internalization of nanoparticles by particle-cell co-localization, but no detectable expression of EGFP. Taken together, our data show that poly (amidoamine)-based nanoparticles can be used for mRNA delivery into cells of the human joint and intervertebral disc, indicating its potential future use as an mRNA delivery system in OA and IVDD, except for macrophages.

Objective: Osteoarthritis (OA) is characterized by articular cartilage erosion, pathological subchondral bone changes, and signs of synovial inflammation and pain. We previously identified p[63-82], a bone morphogenetic protein 7 (BMP7)-derived bioactive peptide that attenuates structural cartilage degeneration in the rat medial meniscal tear-model for posttraumatic OA. This study aimed to evaluate the cartilage erosion-attenuating activity of p[63-82] in a different preclinical model for OA (anterior cruciate ligament transection-partial medial meniscectomy [anterior cruciate ligament transection (ACLT)-pMMx]). The disease-modifying action of the p[63-82] was followed-up in this model for 5 and 10 weeks., Design: Skeletally mature male Lewis rats underwent ACLT-pMMx surgery. Rats received weekly intra-articular injections with either saline or 500 ng p[63-82]. Five and 10 weeks postsurgery, rats were sacrificed, and subchondral bone characteristics were determined using microcomputed tomography (µCT). Histopathological evaluation of cartilage degradation and Osteoarthritis Research Society International (OARSI)-scoring was performed following Safranin-O/Fast Green staining. Pain-related behavior was measured by incapacitance testing and footprint analysis., Results: Histopathological evaluation at 5 and 10 weeks postsurgery showed reduced cartilage degeneration and a significantly reduced OARSI score, whereas no significant changes in subchondral bone characteristics were found in the p[63-82]-treated rats compared to the saline-treated rats. ACLT-pMMx-induced imbalance of static weightbearing capacity in the p[63-82] group was significantly improved compared to the saline-treated rats at weeks 5 postsurgery. Footprint analysis scores in the p[63-82]-treated rats demonstrated improvement at week 10 postsurgery., Conclusions: Weekly intra-articular injections of p[63-82] in the rat ACLT-pMMx posttraumatic OA model resulted in reduced degenerative cartilage changes and induced functional improvement in static weightbearing capacity during follow-up., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: MC and TW are listed as the inventor on filed patents: WO2017178251 and WO2017178253. TW, PE, and LR have shares in Chondropeptix BV.

Introduction: The anterior cruciate ligament (ACL) is susceptible to degeneration, resulting in joint pain, reduced mobility, and osteoarthritis development. There is currently a paucity of knowledge on how anterior cruciate ligament degeneration and disease leads to osteoarthritis. Small non-coding RNAs (sncRNAs), such as microRNAs and small nucleolar RNA (snoRNA), have diverse roles, including regulation of gene expression. Methods: We profiled the sncRNAs of diseased osteoarthritic ACLs to provide novel insights into osteoarthritis development. Small RNA sequencing from the ACLs of non- or end-stage human osteoarthritic knee joints was performed. Significantly differentially expressed sncRNAs were defined, and bioinformatics analysis was undertaken. Results and Discussion: A total of 184 sncRNAs were differentially expressed: 68 small nucleolar RNAs, 26 small nuclear RNAs (snRNAs), and 90 microRNAs. We identified both novel and recognized (miR-206, -365, and -29b and -29c) osteoarthritis-related microRNAs and other sncRNAs (including SNORD72, SNORD113, and SNORD114). Significant pathway enrichment of differentially expressed miRNAs includes differentiation of the muscle, inflammation, proliferation of chondrocytes, and fibrosis. Putative mRNAs of the microRNA target genes were associated with the canonical pathways "hepatic fibrosis signaling" and "osteoarthritis." The establishing sncRNA signatures of ACL disease during osteoarthritis could serve as novel biomarkers and potential therapeutic targets in ACL degeneration and osteoarthritis development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RG declared a shared parent affiliation with the author TW to the handling editor at the time of review., (Copyright © 2023 Kharaz, Zamboulis, Fang, Welting, Peffers and Comerford.)

Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling was analysed using luciferase-based transcription factor activity assays. EV protein cargo appears to be more important during osteoarthritis progression than small non-coding RNAs. Cluster analysis revealed plasma-EVs represented a time-dependent response to osteoarthritis induction associated with supramolecular complexes. Clusters for synovial fluid-derived EVs were associated with initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis for plasma-derived EVs correlated with day post-induction and were primarily composed of proteins modulating lipid metabolism. Synovial fluid-derived EVs factors represented intermediate filament and supramolecular complexes reflecting tissue repair. There was a significant interaction between time and osteoarthritis for CRE, NFkB, SRE, SRF with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect.

Eukaryotic ribosomes are complex molecular nanomachines translating genetic information from mRNAs into proteins. There is natural heterogeneity in ribosome composition. The pseudouridylation (ψ) of ribosomal RNAs (rRNAs) is one of the key sources of ribosome heterogeneity. Nevertheless, the functional consequences of ψ-based ribosome heterogeneity and its relevance for human disease are yet to be understood. Using HydraPsiSeq and a chronic disease model of non-osteoarthritic primary human articular chondrocytes exposed to osteoarthritic synovial fluid, we demonstrated that the disease microenvironment is capable of instigating site-specific changes in rRNA ψ profiles. To investigate one of the identified differential rRNA ψ sites (28S-ψ4966), we generated SNORA22 and SNORA33 KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by 35 S-Met/Cys incorporation, assessed the mode of translation initiation and ribosomal fidelity using dual luciferase reporters, and assessed cellular and ribosomal proteomes by LC-MS/MS. We uncovered that the depletion of SNORA33 , but not SNORA22 , reduced 28S-ψ4966 levels. The resulting loss of 28S-ψ4966 affected ribosomal protein composition and function and led to specific changes in the cellular proteome. Overall, our pioneering findings demonstrate that cells dynamically respond to disease-relevant changes in their environment by altering their rRNA pseudouridylation profiles, with consequences for ribosome function and the cellular proteome relevant to human disease.

Objective: Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods., Method: Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-β) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-β signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors., Results: Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-β signaling, both in activation of latent TGF-β and TGF-β superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-β signaling was confirmed by increased activity of expression of TGF-β target genes and luciferase reporters. Inhibition of BCP driven TGF-β signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression., Conclusion: BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-β signaling was identified in development of a pro-inflammatory environment., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Ectopic calcification is an important contributor to chronic diseases, such as osteoarthritis. Currently, no effective therapies exist to counteract calcification. We developed peptides derived from the calcium binding domain of human Alpha-2-HS-Glycoprotein (AHSG/Fetuin A) to counteract calcification., Methods: A library of seven 30 amino acid (AA) long peptides, spanning the 118 AA Cystatin 1 domain of AHSG, were synthesized and evaluated in an in vitro calcium phosphate precipitation assay. The best performing peptide was modified (cyclic, retro-inverso and combinations thereof) and evaluated in cellular calcification models and the rat Medial Collateral Ligament Transection + Medial Meniscal Tear (MCLT + MMT) osteoarthritis model., Results: A cyclic peptide spanning AA 1-30 of mature AHSG showed clear inhibition of calcium phosphate precipitation in the nM-pM range that far exceeded the biological activity of the linear peptide variant or bovine Fetuin. Biochemical and electron microscopy analyses of calcium phosphate particles revealed a similar, but distinct, mode of action in comparison with bFetuin. A cyclic-inverso variant of the AHSG 1-30 peptide inhibited calcification of human articular chondrocytes, vascular smooth muscle cells and during osteogenic differentiation of bone marrow derived stromal cells. Lastly, we evaluated the effect of intra-articular injection of the cyclic-inverso AHSG 1-30 peptide in a rat osteoarthritis model. A significant improvement was found in histopathological osteoarthritis score and animal mobility. Serum levels of IFNγ were found to be lower in AHSG 1-30 peptide treated animals., Conclusions: The cyclic-inverso AHSG 1-30 peptide directly inhibits the calcification process and holds the potential for future application in osteoarthritis., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Osteoarthritis is the most common degenerative joint disorder. MicroRNAs are gene expression regulators that act post-transcriptionally to control tissue homeostasis. Microarray analysis was undertaken in osteoarthritic intact, lesioned and young intact cartilage. Principal component analysis showed that young intact cartilage samples were clustered together; osteoarthritic samples had a wider distribution; and osteoarthritic intact samples were separated into two subgroups, osteoarthritic-Intact-1 and osteoarthritic-Intact-2. We identified 318 differentially expressed microRNAs between young intact and osteoarthritic lesioned cartilage, 477 between young intact and osteoarthritic-Intact-1 cartilage and 332 between young intact and osteoarthritic-Intact-2 cartilage samples. For a selected list of differentially expressed microRNAs, results were verified in additional cartilage samples using qPCR. Of the validated DE microRNAs, four-miR-107, miR-143-3p, miR-361-5p and miR-379-5p-were selected for further experiments in human primary chondrocytes treated with IL-1β. Expression of these microRNAs decreased in human primary chondrocytes treated with IL-1β. For miR-107 and miR-143-3p, gain- and loss-of-function approaches were undertaken and associated target genes and molecular pathways were investigated using qPCR and mass spectrometry proteomics. Analyses showed that WNT4 and IHH , predicted targets of miR-107, had increased expression in osteoarthritic cartilage compared to young intact cartilage and in primary chondrocytes treated with miR-107 inhibitor, and decreased expression in primary chondrocytes treated with miR-107 mimic, suggesting a role of miR-107 in chondrocyte survival and proliferation. In addition, we identified an association between miR-143-3p and EIF2 signalling and cell survival. Our work supports the role of miR-107 and miR-143-3p in important chondrocyte mechanisms regulating proliferation, hypertrophy and protein translation.

Objective: Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis., Methods: Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation., Results: We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9-14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33-64%]). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI [2.2-4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3-2.0])., Conclusions: Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development., Competing Interests: Declaration of competing interest The authors declare no competing interests that are relevant to the submitted work., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Since the joint microenvironment and tissue homeostasis are highly dependent on synovial fluid, we aimed to compare the essential chondrocyte signaling signatures of non-osteoarthritic vs end-stage osteoarthritic knee synovial fluid. Moreover, we determined the phenotypic consequence of the distinct signaling patterns on articular chondrocytes., Methods: Protein profiling of synovial fluid was performed using antibody arrays. Chondrocyte signaling and phenotypic changes induced by non-osteoarthritic and osteoarthritic synovial fluid were analyzed using a phospho-kinase array, luciferase-based transcription factor activity assays, and RT-qPCR. The origin of osteoarthritic synovial fluid signaling was evaluated by comparing the signaling responses of conditioned media from cartilage, synovium, infrapatellar fat pad and meniscus. Osteoarthritic synovial fluid induced pathway-phenotype relationships were evaluated using pharmacological inhibitors., Results: Compared to non-osteoarthritic synovial fluid, osteoarthritic synovial fluid was enriched in cytokines, chemokines and growth factors that provoked differential MAPK, AKT, NFκB and cell cycle signaling in chondrocytes. Functional pathway analysis confirmed increased activity of these signaling events upon osteoarthritic synovial fluid stimulation. Tissue secretomes of osteoarthritic cartilage, synovium, infrapatellar fat pad and meniscus activated several inflammatory signaling routes. Furthermore, the distinct pathway signatures of osteoarthritic synovial fluid led to accelerated chondrocyte dedifferentiation via MAPK/ERK signaling, increased chondrocyte fibrosis through MAPK/JNK and PI 3 K/AKT activation, an elevated inflammatory response mediated by cPKC/NFκB, production of extracellular matrix-degrading enzymes by MAPK/p38 and PI 3 K/AKT routes, and enabling of chondrocyte proliferation., Conclusion: This study provides the first mechanistic comparison between non-osteoarthritic and osteoarthritic synovial fluid, highlighting MAPKs, cPKC/NFκB and PI 3 K/AKT as crucial OA-associated intracellular signaling routes., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Osteoarthritis (OA) is a degenerative musculoskeletal disorder affecting the whole synovial joint and globally impacts more than one in five individuals aged 40 and over, representing a huge socioeconomic burden. Drug penetration into and retention within the joints are major challenges in the development of regenerative therapies for OA. During the recent years, polymeric nanoparticles (PNPs) have emerged as promising drug carrier candidates due to their biodegradable properties, nanoscale structure, functional versatility, and reproducible manufacturing, which makes them particularly attractive for cartilage penetration and joint retention. In this review, we discuss the current development state of natural and synthetic PNPs for drug delivery and OA treatment. Evidence from in vitro and pre-clinical in vivo studies is used to show how disease pathology and key cellular pathways of joint inflammation are modulated by these nanoparticle-based therapies. Furthermore, we compare the biodegradability and surface modification of these nanocarriers in relation to the drug release profile and tissue targeting. Finally, the main challenges for nanoparticle delivery to the cartilage are discussed, as a function of disease state and physicochemical properties of PNPs such as size and surface charge.

Objective: The potential chondroprotective effect of celecoxib, a nonsteroidal anti-inflammatory drug and selective cyclooxygenase-2 inhibitor used to reduce pain and inflammation in knee osteoarthritis patients, is disputed. This study aimed at investigating the chondroprotective effects of celecoxib on (1) human articular cartilage explants and (2) in an in vivo osteoarthritis rat model., Design: Articular cartilage explants from 16 osteoarthritis patients were cultured for 24 hours with celecoxib or vehicle. Secreted prostaglandins (prostaglandin E 2 , prostaglandin F 2α , prostaglandin D 2 ) and thromboxane B2 (TXB2) concentrations were determined in medium by ELISA, and protein regulation was measured with label-free proteomics. Cartilage samples from 7 of these patients were analyzed for gene expression using real-time quantitative polymerase chain reaction. To investigate the chondroprotective effect of celecoxib in vivo , 14 rats received an intra-articular injection of celecoxib or 0.9% NaCl after osteoarthritis induction by anterior cruciate ligament transection and partial medial meniscectomy (ACLT/pMMx model). Histopathological scoring was used to evaluate osteoarthritis severity 12 weeks after injection., Results: Secretion of prostaglandins, target of Nesh-SH3 (ABI3BP), and osteonectin proteins decreased, whereas tissue inhibitor of metalloproteinase 2 (TIMP-2) increased significantly after celecoxib treatment in the human ( ex vivo ) explant culture. Gene expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS4/5) and metalloproteinase 13 (MMP13) was significantly reduced after celecoxib treatment in human cartilage explants. Cartilage degeneration was reduced significantly in an in vivo osteoarthritis knee rat model., Conclusions: Our data demonstrated that celecoxib acts chondroprotective on cartilage ex vivo and a single intra-articular bolus injection has a chondroprotective effect in vivo .

Objectives: Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients., Materials and Methods: Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation., Results: Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation., Conclusions: Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Ribosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation., Materials: SnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling., Results: As a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5-6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation., Conclusion: The expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases., Competing Interests: None., (© 2022 The Authors.)

BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype., Competing Interests: MMJ Caron and TJM Welting are inventors on patents WO2017178251 and WO2017178253 (Owned by Chondropeptix). LW van Rhijn and TJM Welting are shareholder in Chondropeptix and are CDO, and CSO of Chondropeptix, respectively. All other authors have no competing interests to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Human mesenchymal stromal cells (MSCs) can be used for cartilage repair therapies based on their potential to differentiate into chondrocytes. However, the joint microenvironment is a major determinant of the success of MSC-based cartilage formation. Currently, there is no tool that is able to predict the effect of a patient's OA joint microenvironment on MSC-based cartilage formation. Our goal was to develop a molecular tool that can predict this effect before the start of cartilage repair therapies. Six different promoter reporters (hIL6, hIL8, hADAMTS5, hWISP1, hMMP13, and hADAM28) were generated and evaluated in an immortalized human articular chondrocyte for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm) obtained from 32 different knee OA patients. To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model, while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. We confirmed that OAs-cm of different patients had significantly different effects on sGAG production. In addition, significant correlations were obtained between the effect of the OAs-cm on cartilage formation and promoter reporter outcome. Furthermore, we validated the predictive value of measuring two promoter reporters with an independent cohort of OAs-cm and the effect of 87.5% of the OAs-cm on MSC-based cartilage formation could be predicted. Together, we developed a novel tool to predict the effect of the OA joint microenvironment on MSC-based cartilage formation. This is an important first step toward personalized cartilage repair strategies for OA patients. Impact statement We describe the development of a novel molecular tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. Such a tool is of great importance in determining the success of mesenchymal stromal cell-based cartilage repair strategies.

Purpose of Review: Translation of genetic information encoded within mRNA molecules by ribosomes into proteins is a key part of the central dogma of molecular biology. Despite the central position of the ribosome in the translation of proteins, and considering the major proteomic changes that occur in the joint during osteoarthritis development and progression, the ribosome has received very limited attention as driver of osteoarthritis pathogenesis., Recent Findings: We provide an overview of the limited literature regarding this developing topic for the osteoarthritis field. Recent key findings that connect ribosome biogenesis and activity with osteoarthritis include: ribosomal RNA transcription, processing and maturation, ribosomal protein expression, protein translation capacity and preferential translation., Summary: The ribosome as the central cellular protein synthesis hub is largely neglected in osteoarthritis research. Findings included in this review reveal that in osteoarthritis, ribosome aberrations have been found from early-stage ribosome biogenesis, through ribosome build-up and maturation, up to preferential translation. Classically, osteoarthritis has been explained as an imbalance between joint tissue anabolism and catabolism. We postulate that osteoarthritis can be interpreted as an acquired ribosomopathy. This hypothesis fine-tunes the dogmatic anabolism/katabolism point-of-view, and may provide novel molecular opportunities for the development of osteoarthritis disease-modifying treatments., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Mutations in the non-coding snoRNA component of mitochondrial RNA processing endoribonuclease (RMRP) are the cause of cartilage-hair hypoplasia (CHH). CHH is a rare form of metaphyseal chondrodysplasia characterized by disproportionate short stature and abnormal growth plate development. The process of chondrogenic differentiation within growth plates of long bones is vital for longitudinal bone growth. However, molecular mechanisms behind impaired skeletal development in CHH patients remain unclear. We employed a transdifferentiation model (FDC) combined with whole transcriptome analysis to investigate the chondrogenic transdifferentiation capacity of CHH fibroblasts and to examine pathway regulation in CHH cells during chondrogenic differentiation. We established that the FDC transdifferentiation model is a relevant in vitro model of chondrogenic differentiation, with an emphasis on the terminal differentiation phase, which is crucial for longitudinal bone growth. We demonstrated that CHH fibroblasts are capable of transdifferentiating into chondrocyte-like cells, and show a reduced commitment to terminal differentiation. We also found a number of key factors of BMP, FGF, and IGF-1 signalling axes to be significantly upregulated in CHH cells during the chondrogenic transdifferentiation. Our results support postulated conclusions that RMRP has pleiotropic functions and profoundly affects multiple aspects of cell fate and signalling. Our findings shed light on the consequences of pathological CHH mutations in snoRNA RMRP during chondrogenic differentiation and the relevance and roles of non-coding RNAs in genetic diseases in general., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors.)

Objective: The main purpose of the present study was to assess the risk for major revision surgery after perichondrium transplantation (PT) at a minimum of 22 years postoperatively and to evaluate the influence of patient characteristics., Design: Primary outcome was treatment success or failure. Failure of PT was defined as revision surgery in which the transplant was removed, such as (unicondylar) knee arthroplasty or patellectomy. The functioning of nonfailed patients was evaluated using the International Knee Documentation Committee (IKDC) score. In addition, the influence of patient characteristics was evaluated., Results: Ninety knees in 88 patients, aged 16 to 55 years with symptomatic cartilage defects, were treated by PT. Eighty knees in 78 patients were eligible for analysis and 10 patients were lost to follow-up. Twenty-eight knees in 26 patients had undergone major revision surgery. Previous surgery and a longer time of symptoms prior to PT were significantly associated with an increased risk for failure of cartilage repair. Functioning of the remaining 52 patients and influence of patient characteristics was analyzed using their IKDC score. Their median IKDC score was 39.08, but a relatively young age at transplantation was associated with a higher IKDC score., Conclusions: This 22-year follow-up study of PT, with objective outcome parameters next to patient-reported outcome measurements in a unique group of patients, shows that overall 66% was without major revision surgery and patient characteristics also influence long-term outcome of cartilage repair surgery.

The fibrocartilage chondrocyte phenotype has been recognized to attribute to osteoarthritis (OA) development. These chondrocytes express genes related to unfavorable OA outcomes, emphasizing its importance in OA pathology. BMP7 is being explored as a potential disease-modifying molecule and attenuates the chondrocyte hypertrophic phenotype. On the other hand, BMP7 has been demonstrated to relieve organ fibrosis by counteracting the pro-fibrotic TGFβ-Smad3-PAI1 axis and increasing MMP2-mediated Collagen type I turnover. Whether BMP7 has anti-fibrotic properties in chondrocytes is unknown. Human OA articular chondrocytes (HACs) were isolated from end-stage OA femoral cartilage (total knee arthroplasty; n = 18 individual donors). SW1353 cells and OA HACs were exposed to 1 nM BMP7 for 24 h, after which gene expression of fibrosis-related genes and fibrosis-mediating factors was determined by RT-qPCR. In SW1353, Collagen type I protein levels were determined by immunocytochemistry and western blotting. PAI1 and MMP2 protein levels and activity were measured with an ELISA and activity assays, respectively. MMP2 activity was inhibited with the selective MMP-2 inhibitor OA-Hy. SMAD3 activity was determined by a (CAGA) 12 -reporter assay, and pSMAD2 levels by western blotting. Following BMP7 exposure, the expression of fibrosis-related genes was reduced in SW1353 cells and OA HACs. BMP7 reduced Collagen type I protein levels in SW1353 cells. Gene expression of MMP2 was increased in SW1353 cells following BMP7 treatment. BMP7 reduced PAI1 protein levels and -activity, while MMP2 protein levels and -activity were increased by BMP7. BMP7-dependent inhibition of Collagen type I protein levels in SW1353 cells was abrogated when MMP2 activity was inhibited. Finally, BMP7 reduced pSMAD2 levels determined by western blotting and reduced SMAD3 transcriptional activity as demonstrated by decreased (CAGA) 12 luciferase reporter activity. Our data demonstrate that short-term exposure to BMP7 decreases the fibrocartilage chondrocyte phenotype. The BMP7-dependent reduction of Collagen type I protein expression seems MMP2-dependent and inhibition of Smad2/3-PAI1 activity was identified as a potential pathway via which BMP7 exerts its anti-fibrotic action. This indicates that in chondrocytes BMP7 may have a double mode-of-action by targeting both the hypertrophic as well as the fibrotic chondrocyte phenotype, potentially adding to the clinical relevance of using BMP7 as an OA disease-modifying molecule., (© 2021. The Author(s).)

Objective: Despite the general awareness that cyclo-oxygenase-2 (COX-2) is crucial for endochondral ossification, the role of COX-2 in skeletal development is largely unknown. We hypothesized that inhibition or genetic loss of COX-2 leads to impaired growth plate development and consequently impaired postnatal development of the long bones., Design: Skeletally immature (5 weeks old) B6;129S-Ptgs2 tm1Jed /J wildtype mice were treated for 10 weeks with celecoxib (daily oral administration 10 mg/kg) or placebo and compared with B6;129S-Ptgs2 tm1Jed /J homozygous knockout mice ( n = 12 per group)., Results: Fifteen weeks postnatally, no significant difference in growth plate (zone) thickness was found between groups. However, significantly higher proteoglycan content and lower expression levels of collagen type II and X staining in the growth plates of celecoxib-treated mice, and to a lesser extent in COX-2 knockout mice. In addition, a significantly decreased cell number and cell size were observed in the hypertrophic zone of the growth plates of both experimental groups. Micro-computed tomography analysis of the subchondral bone region directly beneath the growth plate showed significantly higher bone density and trabecular thickness, following celecoxib treatment. Despite the detected differences in growth plate extracellular matrix composition and subchondral bone morphology, no difference was found in the length of the tibia in celecoxib-treated mice or COX-2 knockout mice., Conclusions: Genetic loss of COX-2 or treatment with celecoxib did not result in detectable differences in gross murine formation of the tibia or femur. However, there were notable phenotypic features detected in the maturation of the growth plate (hypertrophic zone and subchondral bone) as a result of the celecoxib treatment.

Introduction: In addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study, we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation., Materials: Sox9 expression in ATDC5 cells was knocked down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 h and 7 days of differentiation. The transcriptomes (RNA-seq approach) and proteomes (Label-free proteomics approach) were compared using pathway and network analyses. Total protein translational capacity was evaluated with the SuNSET assay, active ribosomes were evaluated with polysome profiling, and ribosome modus was evaluated with bicistronic reporter assays., Results: Early Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown., Conclusion: Here, we identified an essential new function for Sox9 during early chondrogenic differentiation. A role for Sox9 in regulation of ribosome amount, activity, and/or composition may be crucial in preparation for the demanding proliferative phase and subsequent cartilage extracellular matrix production of chondroprogenitors in the growth plate in vivo ., Competing Interests: MC and TW are inventor on patents WO2017178251 and WO2017178253 (Licensed to Chondropeptix). LR and TW have shares in Chondropeptix and are CDO and CSO of Chondropeptix. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Caron, Eveque, Cillero-Pastor, Heeren, Housmans, Derks, Cremers, Peffers, van Rhijn, van den Akker and Welting.)

Bicistronic reporter assays have been instrumental for transgene expression, understanding of internal ribosomal entry site (IRES) translation, and identification of novel cap-independent translational elements (CITE). We observed a large methodological variability in the use of bicistronic reporter assays and data presentation or normalization procedures. Therefore, we systematically searched the literature for bicistronic IRES reporter studies and analyzed methodological details, data visualization, and normalization procedures. Two hundred fifty-seven publications were identified using our search strategy (published 1994-2020). Experimental studies on eukaryotic adherent cell systems and the cell-free translation assay were included for further analysis. We evaluated the following methodological details for 176 full text articles: the bicistronic reporter design, the cell line or type, transfection methods, and time point of analyses post-transfection. For the cell-free translation assay, we focused on methods of in vitro transcription, type of translation lysate, and incubation times and assay temperature. Data can be presented in multiple ways: raw data from individual cistrons, a ratio of the two, or fold changes thereof. In addition, many different control experiments have been suggested when studying IRES-mediated translation. In addition, many different normalization and control experiments have been suggested when studying IRES-mediated translation. Therefore, we also categorized and summarized their use. Our unbiased analyses provide a representative overview of bicistronic IRES reporter use. We identified parameters that were reported inconsistently or incompletely, which could hamper data reproduction and interpretation. On the basis of our analyses, we encourage adhering to a number of practices that should improve transparency of bicistronic reporter data presentation and improve methodological descriptions to facilitate data replication.

Treatment of osteoarthritis (OA) is mainly symptomatic by alleviating pain to postpone total joint replacement. Bone morphogenetic protein 7 (BMP7) is a candidate morphogen for experimental OA treatment that favorably alters the chondrocyte and cartilage phenotype. Intra-articular delivery and sustained release of a recombinant growth factor for treating OA are challenging, whereas the use of peptide technology potentially circumvents many of these challenges. In this study, we screened a high-resolution BMP7 peptide library and discovered several overlapping peptide sequences from two regions in BMP7 with nanomolar bioactivity that attenuated the pathological OA chondrocyte phenotype. A single exposure of OA chondrocytes to peptides p[63-82] and p[113-132] ameliorated the OA chondrocyte phenotype for up to 8 days, and peptides were bioactive on chondrocytes in OA synovial fluid. Peptides p[63-82] and p[113-132] required NKX3-2 for their bioactivity on chondrocytes and provoke changes in SMAD signaling activity. The bioactivity of p[63-82] depended on specific evolutionary conserved sequence elements common to BMP family members. Intra-articular injection of a rat medial meniscal tear (MMT) model with peptide p[63-82] attenuated cartilage degeneration. Together, this study identified two regions in BMP7 from which bioactive peptides are able to attenuate the OA chondrocyte phenotype. These BMP7-derived peptides provide potential novel disease-modifying treatment options for OA., Competing Interests: The study sponsors had no involvement in study design, collection, analysis, and interpretation of data; the writing of the manuscript; or the decision to submit the manuscript for publication. M.M.J.C. and T.J.M.W. are inventors on patents WO2017178251 and WO2017178253 (owned by Chondropeptix). P.J.E., L.W.v.R., and T.J.M.W. are shareholders in Chondropeptix and are CMO, CDO, and CSO of Chondropeptix, respectively. The other authors declare no competing interests., (© 2021 The Authors.)

Restriction of physical growth and development is a known problem in patients with juvenile idiopathic arthritis (JIA). However, the effect of medical treatment for JIA on skeletal growth in affected children has not been properly investigated. We, therefore, hypothesize that naproxen and methotrexate (MTX) affect endochondral ossification and will lead to reduced skeletal development. Treatment of ATDC5 cells, an in vitro model for endochondral ossification, with naproxen or MTX resulted in increased chondrogenic but decreased hypertrophic differentiation. In vivo, healthy growing C57BL/6 mice were treated with naproxen, MTX, or placebo for 10 weeks. At 15 weeks postnatal, both the length of the tibia and the length of the femur were significantly reduced in the naproxen- and MTX-treated mice compared to their controls. Growth plate analysis revealed a significantly thicker proliferative zone, while the hypertrophic zone was significantly thinner in both experimental groups compared to their controls, comparable to the in vitro results. Micro-computed tomography analysis of the subchondral bone region directly below the growth disc showed significantly altered bone microarchitecture in naproxen and MTX groups. In addition, the involvement of the PTHrP-Ihh loop in naproxen- and MTX-treated cells was shown. Overall, these results demonstrate that naproxen and MTX have a profound effect on endochondral ossification during growth plate development, abnormal subchondral bone morphology, and reduced bone length. A better understanding of how medication influences the development of the growth plate will improve understanding of endochondral ossification and reveal possibilities to improve the treatment of pediatric patients., (© 2020 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Reporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we compared the characteristics of five reporter genes (Firefly luciferase, stable- and unstable Nano luciferase, secretable Gaussia luciferase and Red Fluorescent Protein) to study complex body fluids. For this comparison, the NFκB Response Element (NFκB-RE) and Smad Binding Element (SBE) were identically cloned into the five different reporter vectors. Reporter characteristics were evaluated by kinetic and concentration-response measurements in SW1353 and HeLa cell lines. Finally, reporter compatibility with complex body fluids (fetal calf serum, knee joint synovial fluid and human serum) and inter-donor variation were evaluated. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases. Intracellularly measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. Secreted Gaussia luciferase appeared to be incompatible with complex body fluids, due to variability in inter-donor signal interference. Unstable Nano luciferase demonstrated clear inducibility, high sensitivity and compatibility with complex body fluids and therefore can be recommended for cellular signaling studies using complex body fluids.

Background: Osteoarthritis remains one of the greatest causes of morbidity and mortality in the equine population. The inability to detect pre-clinical changes in osteoarthritis has been a significant impediment to the development of effective therapies against this disease. Synovial fluid represents a potential source of disease-specific small non-coding RNAs (sncRNAs) that could aid in the understanding of the pathogenesis of osteoarthritis. We hypothesised that early stages of osteoarthritis would alter the expression of sncRNAs, facilitating the understanding of the underlying pathogenesis and potentially provide early biomarkers., Methods: Small RNA sequencing was performed using synovial fluid from the metacarpophalangeal joints of both control and early osteoarthritic horses. A group of differentially expressed sncRNAs was selected for further validation through qRT-PCR using an independent cohort of synovial fluid samples from control and early osteoarthritic horses. Bioinformatic analysis was performed in order to identify putative targets of the differentially expressed microRNAs and to explore potential associations with specific biological processes., Results: Results revealed 22 differentially expressed sncRNAs including 13 microRNAs; miR-10a, miR-223, let7a, miR-99a, miR-23b, miR-378, miR-143 (and six novel microRNAs), four small nuclear RNAs; U2, U5, U11, U12, three small nucleolar RNAs; U13, snoR38, snord96, and one small cajal body-specific RNA; scarna3. Five sncRNAs were validated; miR-223 was significantly reduced in early osteoarthritis and miR-23b, let-7a-2, snord96A and snord13 were significantly upregulated. Significant cellular actions deduced by the differentially expressed microRNAs included apoptosis (P < 0.0003), necrosis (P < 0.0009), autophagy (P < 0.0007) and inflammation (P < 0.00001). A conservatively filtered list of 57 messenger RNA targets was obtained; the top biological processes associated were regulation of cell population proliferation (P < 0.000001), cellular response to chemical stimulus (P < 0.000001) and cell surface receptor signalling pathway (P < 0.000001)., Conclusions: Synovial fluid sncRNAs may be used as molecular biomarkers for early disease in equine osteoarthritic joints. The biological processes they regulate may play an important role in understanding early osteoarthritis pathogenesis. Characterising these dynamic molecular changes could provide novel insights on the process and mechanism of early osteoarthritis development and is critical for the development of new therapeutic approaches.

Cells isolated from the intervertebral disc are often used for in vitro experimentation. Correctly separating the intervertebral disc tissue in annulus fibrosus and nucleus pulposus is particularly challenging when working with surplus material from surgery or specimens from donors with an advanced age. Moreover, lineage controls are only sparsely reported to verify tissue of origin. Here we describe an approach to intervertebral disc cell isolation from human and bovine origin.

Osteoarthritis (OA) presents as a change in the articular chondrocyte phenotype. The origin of the phenotype change is poorly understood. Small nucleolar RNAs (snoRNAs) direct chemical modification of other RNA substrates and are involved in endoribonucleolytic pre-rRNA processing. They have thereby a role by fine-tuning spliceosome and ribosome function and can thus accommodate changing requirements for protein synthesis in OA. Here we describe both targeted and global methods for snoRNA isolation and quantification from whole cartilage.

Objective: For many proteins from osteoarthritic synovial fluid, their intra-articular tissue of origin remains unknown. In this study we performed comparative proteomics to identify osteoarthritis-specific and joint tissue-dependent secreted proteins that may serve as candidates for osteoarthritis biomarker development on a tissue-specific basis., Design: Protein secretomes of cartilage, synovium, Hoffa's fat pad and meniscus from knee osteoarthritis patients were determined using liquid chromatography tandem mass spectrometry, followed by label-free quantification. Validation of tissue-dependent protein species was conducted by ELISA on independent samples. Differential proteomes of osteoarthritic and non-osteoarthritic knee synovial fluids were obtained via similar proteomics approach, followed by ELISA validation., Results: Proteomics revealed 64 proteins highly secreted from cartilage, 94 from synovium, 37 from Hoffa's fat pad and 21 from meniscus. Proteomic analyses of osteoarthritic vs non-osteoarthritic knee synovial fluid revealed 70 proteins with a relatively higher abundance and 264 proteins with a relatively lower abundance in osteoarthritic synovial fluid. Of the 70 higher abundance proteins, 23 were amongst the most highly expressed in the secretomes of a specific intra-articular tissue measured. Tissue-dependent release was validated for SLPI, C8, CLU, FN1, RARRES2, MATN3, MMP3 and TNC. Abundance in synovial fluid of tissue-dependent proteins was validated for IGF2, AHSG, FN1, CFB, KNG and C8., Conclusions: We identified proteins with a tissue-dependent release from intra-articular human knee OA tissues. A number of these proteins also had an osteoarthritis-specific abundance in knee synovial fluid. These proteins may serve as novel candidates for osteoarthritis biomarker development on a tissue-specific basis., Competing Interests: Conflict of Interests TJM Welting is listed as inventor on patents: WO2017178251, WO2017178253 and US 20130123314. PJ Emans and LW van Rhijn are listed as inventors on patent US 20130123314. LW van Rhijn, PJ Emans and TJM Welting have shares in Chondropeptix and are CDO, CMO and CSO of Chondropeptix, respectively., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Focal knee resurfacing implants (FKRIs) are intended to treat cartilage defects in middle-aged patients. Most FKRIs are metal-based, which hampers follow-up of the joint using magnetic resonance imaging and potentially leads to damage of the opposing cartilage. The purpose of this study was to develop a nondegradable thermoplastic polyurethane (TPU) FKRI and investigate its osseointegration. Different surface roughness modifications and biphasic calcium phosphate (BCP) coating densities were first tested in vitro on TPU discs. The in vivo osseointegration of BCP-coated TPU implants was subsequently compared to uncoated TPU implants and the titanium bottom layer of metal control implants in a caprine model. Implants were implanted bilaterally in stifle joints and animals were followed for 12 weeks, after which the bone-to-implant contact area (BIC) was assessed. Additionally, 18F-sodium-fluoride (18F-NaF) positron emission tomography PET/CT-scans were obtained at 3 and 12 weeks to visualize the bone metabolism over time. The BIC was significantly higher for the BCP-coated TPU implants compared to the uncoated TPU implants (p = .03), and did not significantly differ from titanium (p = .68). Similar 18F-NaF tracer uptake patterns were observed between 3 and 12 weeks for the BCP-coated TPU and titanium implants, but not for the uncoated implants. TPU FKRIs with surface modifications could provide the answer to the drawbacks of metal FKRIs., (© 2020 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

Knee osteoarthritis (OA) is a condition mainly characterized by cartilage degradation. Currently, no effective treatment exists to slow down the progression of OA-related cartilage damage. Selective COX-2 inhibitors may, next to their pain killing properties, act chondroprotective in vivo. To determine whether the route of administration is important for the efficacy of the chondroprotective properties of selective COX-2 inhibitors, a systematic review was performed according to the PRISMA guidelines. Studies investigating OA-related cartilage damage of selective COX-2 inhibitors in vivo were included. Nine of the fourteen preclinical studies demonstrated chondroprotective effects of selective COX-2 inhibitors using systemic administration. Five clinical studies were included and, although in general non-randomized, failed to demonstrate chondroprotective actions of oral selective COX-2 inhibitors. All of the four preclinical studies using bolus intra-articular injections demonstrated chondroprotective actions, while one of the three preclinical studies using a slow release system demonstrated chondroprotective actions. Despite the limited evidence in clinical studies that have used the oral administration route, there seems to be a preclinical basis for considering selective COX-2 inhibitors as disease modifying osteoarthritis drugs when used intra-articularly. Intra-articularly injected selective COX-2 inhibitors may hold the potential to provide chondroprotective effects in vivo in clinical studies.

The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by hypertrophic differentiation of the engineered cartilaginous tissue caused by endochondral ossification. Since a healthy cartilage matrix contains high amounts of Aggrecan and COMP, we hypothesized that their supplementation in the biogel used in the generation of subperiosteal cartilage mimics the composition of the cartilage extracellular matrix environment, with beneficial properties for the engineered cartilage. Supplementation of COMP or Aggrecan was studied in vitro during chondrogenic differentiation of rabbit periosteum cells and periosteum-derived chondrocytes. Low melting agarose was supplemented with bovine Aggrecan, human recombinant COMP or vehicle and was injected between the bone and periosteum at the upper medial side of the tibia of New Zealand white rabbits. Generated subperiosteal cartilage tissue was analyzed for weight, GAG and DNA content and ALP activity. Key markers of different phases of endochondral ossification were measured by RT-qPCR. For the in vitro experiments, no significant differences in chondrogenic marker expression were detected following COMP or Aggrecan supplementation, while in vivo favorable chondrogenic marker expression was detected. Gene expression levels of hypertrophic markers as well as ALP activity were significantly decreased in the Aggrecan and COMP supplemented conditions compared to controls. The wet weight and GAG content of the in vivo generated subperiosteal cartilage tissue was not significantly different between groups. Data demonstrate the potential of Aggrecan and COMP to favorably influence the subperiosteal microenvironment for the in vivo generation of cartilage for the optimization of cartilage regenerative approaches., (Copyright © 2020 Caron, Janssen, Peeters, Haudenschild, Cremers, Surtel, van Rhijn, Emans and Welting.)

Although pathways controlling ribosome activity have been described to regulate chondrocyte homeostasis in osteoarthritis, ribosome biogenesis in osteoarthritis is unexplored. We hypothesized that U3 snoRNA, a non-coding RNA involved in ribosomal RNA maturation, is critical for chondrocyte protein translation capacity in osteoarthritis. U3 snoRNA was one of a number of snoRNAs with decreased expression in osteoarthritic cartilage and osteoarthritic chondrocytes. OA synovial fluid impacted U3 snoRNA expression by affecting U3 snoRNA gene promoter activity, while BMP7 was able to increase its expression. Altering U3 snoRNA expression resulted in changes in chondrocyte phenotype. Interference with U3 snoRNA expression led to reduction of rRNA levels and translational capacity, whilst induced expression of U3 snoRNA was accompanied by increased 18S and 28S rRNA levels and elevated protein translation. Whole proteome analysis revealed a global impact of reduced U3 snoRNA expression on protein translational processes and inflammatory pathways. For the first time we demonstrate implications of a snoRNA in osteoarthritis chondrocyte biology and investigated its role in the chondrocyte differentiation status, rRNA levels and protein translational capacity.

Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.

Osteoarthritis presents as a change in the chondrocyte phenotype and an imbalance between anabolic and catabolic processes. Age affects its onset and progression. Small nucleolar RNAs (SnoRNAs) direct chemical modification of RNA substrates to fine-tune spliceosomal and rRNA function, accommodating changing requirements for splicing and protein synthesis during health and disease. Articular cartilage from young, old and OA knees was used in a microarray study to identify alterations in snoRNA expression. Changes in snoRNAs in osteoarthritis-like conditions were studied in chondrocytes using interleukin-1 and osteoarthritic synovial fluid. SNORD26 and SNORD96A knockdown and overexpression were undertaken using antisense oligonucleotides and overexpression plasmids. We identified panels of snoRNAs differentially expressed due to ageing (including SNORD96A, SNORD44) and osteoarthritis (including SNORD26 and SNORD116). In vitro experiments using osteoarthritis-like conditions affected snoRNA expression. Knockdown or overexpression of SNORD26 or SNORD96A resulted in changes in chondrogenic, hypertrophic, rRNA and osteoarthritis related gene expression. We demonstrate that snoRNA expression changes in cartilage ageing, and osteoarthritis and in osteoarthritis-like conditions, and when the expression of these snoRNAs is altered this affects chondrogenic and hypertrophic gene expression. Thus, we propose an additional dimension in the molecular mechanisms underlying cartilage ageing and osteoarthritis through the dysregulation of snoRNAs.

Objective: Lack of specific marker-sets prohibits definition and functional distinction of cellular subtypes in the intervertebral disc (IVD), such as those from the annulus fibrosus (AF) and the nucleus pulposus (NP)., Design: We recently generated immortalized cell lines from human NP and AF tissues; these comprise a set of functionally distinct clonal subtypes. Whole transcriptome analyses were performed of 12 phenotypically distinct clonal cell lines (4× NP-Responder, 4× NP-nonResponder, 2× AF-Sheet forming, and 2× AF-nonSheet forming). Data sets were filtered for membrane-associated marker genes and compared to literature., Results: Comparison of our immortal cell lines to published primary NP, AF, and articular chondrocytes (AC) transcriptome datasets revealed preservation of AF and NP phenotypes. NP-specific membrane-associated genes were defined by comparison to AF cells in both the primary dataset (46 genes) and immortal cell-lines (161 genes). Definition of AF-specific membrane-associated genes yielded 125 primary AF cell and 92 immortal cell-line markers. Overlap between primary and immortal NP cells yielded high-confidence NP-specific marker genes for NP-R ( CLDN11, TMEFF2, CA12, ANXA2, CD44 ) and NP-nR (EFNA1, NETO2, SLC2A1). Overlap between AF and immortal AF subtypes yielded specific markers for AF-S ( COLEC12, LPAR1 ) and AF-nS ( CHIC1 )., Conclusions: The current study provides a reference platform for preclinical evaluation of novel membrane-associated cell type-specific markers in the IVD. Future research will focus on their biological relevance for IVD function in development, homeostasis, and degenerate conditions.

Frank-Ter Haar syndrome (FTHS), Winchester syndrome (WS), and multicentric osteolysis, nodulosis, and arthropathy (MONA) are ultra-rare multisystem disorders characterized by craniofacial malformations, reduced bone density, skeletal and cardiac anomalies, and dermal fibrosis. These autosomal recessive syndromes are caused by homozygous mutation or deletion of respectively SH3PXD2B (SH3 and PX Domains 2B), MMP14 (matrix metalloproteinase 14), or MMP2. Here, we give an overview of the clinical features of 63 previously reported patients with an SH3PXD2B, MMP14, or MMP2 mutation, demonstrating considerable clinical overlap between FTHS, WS, and MONA. Interestingly, the protein products of SH3PXD2B, MMP14, and MMP2 directly cooperate in collagen remodeling. We review animal models for these three disorders that accurately reflect the major clinical features and likewise show significant phenotypical similarity with each other. Furthermore, they demonstrate that defective collagen remodeling is central in the underlying pathology. As such, we propose a nosological revision, placing these SH3PXD2B, MMP14, and MMP2 related syndromes in a novel "defective collagen-remodelling spectrum (DECORS)". In our opinion, this revised nosology better reflects the central role for impaired collagen remodeling, a potential target for pharmaceutical intervention., (© 2019 Wiley Periodicals, Inc.)

Viperin (also known as radical SAM domain-containing 2 (RSAD2)) is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses. Viperin mRNA is a substrate for endoribonucleolytic cleavage by RNase mitochondrial RNA processing (MRP) and mutations in the RNase MRP small nucleolar RNA (snoRNA) subunit of the RNase MRP complex cause cartilage-hair hypoplasia (CHH), a human developmental condition characterized by metaphyseal chondrodysplasia and severe dwarfism. It is unknown how CHH-pathogenic mutations in RNase MRP snoRNA interfere with skeletal development, and aberrant processing of RNase MRP substrate RNAs is thought to be involved. We hypothesized that viperin plays a role in chondrogenic differentiation. Using immunohistochemistry, real-time quantitative PCR, immunoblotting, ELISA, siRNA-mediated gene silencing, plasmid-mediated gene overexpression, label-free MS proteomics, and promoter reporter bioluminescence assays, we discovered here that viperin is expressed in differentiating chondrocytic cells and regulates their protein secretion and the outcome of chondrogenic differentiation by influencing transforming growth factor β (TGF-β)/SMAD family 2/3 (SMAD2/3) activity via C- X -C motif chemokine ligand 10 (CXCL10). Of note, we observed disturbances in this viperin-CXCL10-TGF-β/SMAD2/3 axis in CHH chondrocytic cells. Our results indicate that the antiviral protein viperin controls chondrogenic differentiation by influencing secretion of soluble proteins and identify a molecular route that may explain impaired chondrogenic differentiation of cells from individuals with CHH., (© 2019 Steinbusch et al.)

Despite osteoarthritis (OA) and rheumatoid arthritis (RA) being typically age-related, their underlying etiologies are markedly different. We used 1 H nuclear magnetic resonance (NMR) spectroscopy to identify differences in metabolite profiles in low volumes of OA and RA synovial fluid (SF). SF was aspirated from knee joints of 10 OA and 14 RA patients. 100 μL SF was analyzed using a 700 MHz Avance IIIHD Bruker NMR spectrometer with a TCI cryoprobe. Spectra were analyzed by Chenomx, Bruker TopSpin and AMIX software. Statistical analysis was undertaken using Metaboanalyst. 50 metabolites were annotated, including amino acids, saccharides, nucleotides and soluble lipids. Discriminant analysis identified group separation between OA and RA cohorts, with 32 metabolites significantly different between OA and RA SF (false discovery rate (FDR) < 0.05). Metabolites of glycolysis and the tricarboxylic acid cycle were lower in RA compared to OA; these results concur with higher levels of inflammation, synovial proliferation and hypoxia found in RA compared to OA. Elevated taurine in OA may indicate increased subchondral bone sclerosis. We demonstrate that quantifiable differences in metabolite abundance can be measured in low volumes of SF by 1 H NMR spectroscopy, which may be clinically useful to aid diagnosis and improve understanding of disease pathogenesis.