Background: Synovial inflammation is associated with pain severity in patients with knee osteoarthritis (OA). The aim here was to determine in a population with knee OA, whether synovial tissue from areas associated with pain exhibited different synovial fibroblast subsets, compared to synovial tissue from sites not associated with pain. A further aim was to compare differences between early and end-stage disease synovial fibroblast subsets. Methods: Patients with early knee OA (n = 29) and end-stage knee OA (n = 22) were recruited. Patient reported pain was recorded by questionnaire and using an anatomical knee pain map. Proton density fat suppressed MRI axial and sagittal sequences were analysed and scored for synovitis. Synovial tissue was obtained from the medial and lateral parapatellar and suprapatellar sites. Fibroblast single cell RNA sequencing was performed using Chromium 10X and analysed using Seurat. Transcriptomes were functionally characterised using Ingenuity Pathway Analysis and the effect of fibroblast secretome on neuronal growth assessed using rat DRGN. Findings: Parapatellar synovitis was significantly associated with the pattern of patient-reported pain in knee OA patients. Synovial tissue from sites of patient-reported pain exhibited a differential transcriptomic phenotype, with distinct synovial fibroblast subsets in early OA and end-stage OA. Functional pathway analysis revealed that synovial tissue and fibroblast subsets from painful sites promoted fibrosis, inflammation and the growth and activity of neurons. The secretome of fibroblasts from early OA painful sites induced greater survival and neurite outgrowth in dissociated adult rodent dorsal root ganglion neurons. Interpretation: Sites of patient-reported pain in knee OA exhibit a different synovial tissue phenotype and distinct synovial fibroblast subsets. Further interrogation of these fibroblast pathotypes will increase our understanding of the role of synovitis in OA joint pain and provide a rationale for the therapeutic targeting of fibroblast subsets to alleviate pain in patients.

Background Osteoarthritis (OA), a multifaceted condition, poses a significant challenge for the successful clinical development of therapeutics due to heterogeneity. However, classifying molecular endotypes of OA pathogenesis could provide invaluable phenotype‐directed routes for stratifying subgroups of patients for targeted therapeutics, leading to greater chances of success in trials. This study establishes endotypes in OA soft joint tissue driven by obesity in both load‐bearing and non‐load bearing joints. Methods Hand, hip, knee and foot joint synovial tissue was obtained from OA patients ( n = 32) classified as obese (BMI > 30) or normal weight (BMI 18.5–24.9). Isolated fibroblasts (OA SF) were assayed by Olink proteomic panel, seahorse metabolic flux assay, Illumina's NextSeq 500 bulk and Chromium 10X single cell RNA‐sequencing, validated by Luminex and immunofluorescence. Results Targeted proteomic, metabolic and transcriptomic analysis found the inflammatory landscape of OA SFs are independently impacted by obesity, joint loading and anatomical site with significant heterogeneity between obese and normal weight patients, confirmed by bulk RNAseq. Further investigation by single cell RNAseq identified four functional molecular endotypes including obesity specific subsets defined by an inflammatory endotype related to immune cell regulation, fibroblast activation and inflammatory signaling, with up‐regulated CXCL12, CFD and CHI3L1 expression. Luminex confirmed elevated chitase3‐like‐1(229.5 vs. 49.5 ng/ml, p < .05) and inhibin (20.6 vs. 63.8 pg/ml, p < .05) in obese and normal weight OA SFs, respectively. Lastly, we find SF subsets in obese patients spatially localise in sublining and lining layers of OA synovium and can be distinguished by differential expression of the transcriptional regulators MYC and FOS. Conclusion These findings demonstrate the significance of obesity in changing the inflammatory landscape of synovial fibroblasts in both load bearing and non‐load bearing joints. Describing multiple heterogeneous OA SF populations characterised by specific molecular endotypes, which drive heterogeneity in OA disease pathogenesis. These molecular endotypes may provide a route for the stratification of patients in clinical trials, providing a rational for the therapeutic targeting of specific SF subsets in specific patient populations with arthritic conditions.

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