Knee osteoarthritis (OA) is characterized by chronic, progressive degeneration impacting the entire joint tissue, often leading to total knee arthroplasty in the end stage. Understanding the pathological progression and identifying early intervention biomarkers is crucial to alleviating patient burden. This study focuses on Hoffa's fat pad (HFP), an adipose tissue in the knee's anterior compartment, known to influence OA inflammatory processes. While HFP's role in OA has been recognized, its potential in diagnosing or predicting disease progression is underexplored. Our research aims to bridge this gap by conducting a comparative proteomics analysis of HFP samples from patients with cartilage defects (CD) and those with end-stage OA, seeking to identify sensitive and specific protein alterations associated with OA progression.

In this study, we analyzed HFP samples from 7 CD patients and 22 OA patients. OA patients were divided into 3 subgroups, including 10 non-obese, 8 obese, 4 diabetes. The HFP samples, collected postoperatively, underwent tissue homogenization, protein extraction, and single‐pot solid‐phase‐enhanced sample preparation on a Bravo robotic platform. We employed a multi-proteomics approach, integrating label-free DIA-based quantitative proteomics for extensive HFP proteome profiling and Olink proximity extension assay for targeted quantification of 92 cardiometabolic biomarkers. Statistical analysis was performed using one-way ANOVA analysis, with age factor correction and false discovery rate correction. Pathway enrichment analysis was performed in Cytoscape.

Our study identified elevated levels of 28 proteins in all OA subgroups compared to CD patients (Fig 1.A). These proteins primarily function in binding and catalytic activity. Notably, the increased level of TGFBI was consistent between MS and Olink measurements. TGFBI, an extracellular matrix protein, is an important regulator of cartilage homeostasis. Additionally, other consistent proteins, including CRTAC1 and VASN, showed increased levels in non-obese OA. VCAM1 showed increased levels in diabetic OA (Fig 1.B). Previous studies have highlighted the promising role of CRTAC1 as a specific predictor of OA progression to joint replacement. Our results demonstrated its first-time deregulation in HFP and its association with OA. Pathway analysis showed significant changes in energy exploitation and immune response across all OA endotypes. (Fig 1.C).

Our study leveraged a multi-proteomics approach and demonstrated the capability of HFP in identifying biomarkers that delineate the progression from CD to OA marked by chronic inflammation and degeneration. The alterations in key proteins like CRTCA1 and VCAM1 align with previous serum studies, suggesting a potential role of HFP in mediating their secretion within the joint environment. Future research will validate these potential markers and their expressions using tissue immunohistochemistry and fresh HFP secretome analysis.