A promising therapy for early osteoarthritis (OA) is the transplantation of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs). The synovial fluid (SF) from a pre-clinical ovine model treated with hUC-MSCs has been profiled using proteomics and bioinformatics to elucidate potential mechanisms of therapeutic effect. Four weeks after a medial meniscus transection surgery, sheep were injected with 107 hUC-MSCs in Phosphate Buffered Saline (PBS) or PBS only (n=7) and sacrificed at 12 weeks. SF was normalised for protein abundance (ProteoMinerTM) and analysed using label-free quantitation proteomics. Bioinformatics analyses (Ingenuity Pathway Analysis (IPA) and STRING) were used to assess differentially regulated functions from the proteomic data. Human orthologues were identified for the ovine proteins using UniProt and DAVID resources and proteins that were ≥±1.3 fold differentially abundant between treatment groups, were included in the bioinformatics analyses.Abstract
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Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates. The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture. Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes. RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment. RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes.
Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA.
In a recent phase 2 superiority clinical trial we demonstrated that a single dose of 60mg of the human monoclonal antibody denosumab inhibits osteolytic lesion activity in patients undergoing revision total hip arthroplasty (THA), demonstrating proof of biological efficacy for this clinical application. Here, we examined the effect that denosumab has on disease biology at the osteolysis tissue level. Osteolytic tissue taken from the prosthesis-bone lesion interface at revision surgery in patients with osteolysis (n=10 participants that had received a single 60 mg dose of denosumab 8 weeks prior to revision surgery and n=10 that had received placebo) was examined for total genetic message activity and protein levels using whole genome sequencing and mass spectrometry, respectively. The top five upregulated enriched pathways with denosumab treatment included inflammatory response, myeloid cell activation, myeloid leukocyte migration, neutrophil and granulocyte activation (p<6.26 × 10−28). Cell morphogenesis was amongst the most downregulated pathways (p<3.42 ×10−23). Finally, comparison of the trial mRNA and protein data versus mouse single cell RNA sequencing data of the same pathway blockade in mouse tibia showed the same direction of effect, suggesting that giving the drug causes then cells responsible for osteolysis to disperse into a more immature form (128 of 189 genes (z=4.87, P<0.0001) disease and functional pathways at the mRNA level and 10 of 11 (z=2.72, P=0.0065) at the protein level). In this first-in-man study we identify multiple genes and pathways within periprosthetic osteolysis tissue that are affected by denosumab treatment. The dominant pathways involved upregulation of innate inflammatory signaling and downregulation of cell morphogenesis. We also found enrichment of similar disease and functional pathways at both the mRNA and protein levels versus mRNA pathway enrichment found in mouse osteomorphs. These data provide the first human data of the mechanistic effect of denosumab treatment on inflammatory osteolytic lesion activity after joint replacement that is necessary to support its clinical application. ∗Winner of The Bone & Joint Journal prize∗
Proteomic analysis has the ability to reveal both the different types and abundances of proteins in a sample. To date, proteomic analysis has received limited attention in the field of tendon research, with mainly Six microdialysis samples were obtained from human subjects before (controls) or after shock wave therapy on their achilles tendon. Samples were concentrated and intefering substances removed using StrataClean™ resin. Reduction, alkylation and an In-solution tryptic digestion was performed with the prior addition of 1% Rapigest SF solution. Samples were then analysed by Liquid Chromatography Mass Spectrometry/Mass Spectrometry. Data files were searched using IPI-human database using Mascot Search Engine. Relative quantification was performed between groups by ProgenesisQI.Introduction
Materials and Methods
