Progression of Ankle Osteoarthritis Is Associated with Distinct Synovial Phenotypes
Authors: Matthias Julia , David Michael A. , Buckley Sara E. , Zona Nathaniel , Zuscik Michael , Hunt Kenneth 
Published: December 2024

Category: Ankle Arthritis; Basic Sciences/Biologics Introduction/Purpose: Despite the high incidence and socioeconomic burden of ankle osteoarthritis (OA), limited research has been undertaken to identify factors driving the ankle-specific disease process. While research mainly focuses on cartilage as the place where osteoarthritic combat is fought, little is known about the contribution of synovium throughout the lengthy process of joint degeneration despite its proximity to the joint battlefield. We recently uncovered a unique transcriptomic and histologic signature in ankle OA synovium compared to the knee, suggesting a distinctive synovial disease process in ankle OA. Thus, this study aimed to characterize ankle synovium's transcriptomic and histological landscape with progression of ankle OA. Methods: Synovial samples from patients undergoing routine ankle procedures (IRB-approved) were harvested for bulk RNA sequencing and digital histopathological (DH) analysis. Standard Gene Ontology (GO) analysis was used to identify enriched gene pathways. Synovium was assigned to an OA severity group based on Outerbridge grade (OB) as either minimal (OB-0/1, n=8), moderate (OB-2/3, n=13), and end-stage (OB-4, n=29) OA (Fig 1A). Paraffin-embedded ankle synovium was sectioned and stained with Safranin-O and Picro-Sirius-Red to identify cells, proteoglycans, and collagens, respectively. Sections were imaged using a slide scanner and imported into QuPath software for quantitative and qualitative analysis. Picro-Sirius-Red sections were imaged under polarized light and imported into Python programming to quantify the extent of thin (green color channel)- vs thick (red color channel)-fibered collagenous tissue. Differences for each histological metric were determined using unpaired t-tests with statistical and trending significance set at p < 0.05 and 0.05< p< 0.10, respectively. Results: While moderate disease is characterized by GO terms related to endothelial chemotaxis, vascular growth factor binding and PI3K signaling, the most significant changes were an increase of osteogenic and chondrogenic gene transcripts at end-stage ankle OA (Fig. 1B). Specifically, Col2A1 and 5 immunoglobulin components were among the top upregulated genes. GO terms also showed a decreased expression of pathways involved in mRNA translation, post-transcriptional modification, and ribosome assembly (Fig. 1B). Safranin-O staining showed diffuse proteoglycan deposition in advanced ankle OA, primarily around areas of loose connective tissue with increased vascularization (Fig. 2C). Picro-Sirius-Red staining showed an increased intensity and surface area of thin, but not thick, collagen fibers in advanced ankle OA (Fig. 2D). Conclusion: Synovial phenotypes with progressive ankle OA were distinct based on molecular and tissue-level analyses (Fig 1E). While early disease was characterized by tissue remodeling, late disease showed an inflammatory signature with activation of osteo-chondrogenic pathways. Interestingly, end-stage OA revealed a reduction of transcripts involved in protein production. As proteins are the building blocks of cells and tissues, further research is needed to understand the impact of this molecular signature on the phenotype osteoarthritic synovium. A clear understanding of cellular and transcriptional dynamics of the early osteoarthritic synovium will guide the development of targeted treatment and prevention strategies for ankle OA.