High-Resolution N-Glycan MALDI Mass Spectrometry Imaging of Subchondral Bone Tissue Microarrays in Patients with Knee Osteoarthritis

Abstract

N-glycan alterations contribute to the progression of several joint diseases, including knee osteoarthritis (KOA). However, molecular changes in KOA subchondral trabecular bone, when exposed to different joint loading forces, are still unknown. The aim of this study was, therefore, to demonstrate the feasibility to differentiate Nglycan changes in subchondral trabecular bone from four different joint loading forces of the tibial plateau regions (i.e., Lateral Anterior (L-A), Lateral Posterior (L-P), Medial Anterior (M-A), and Medial Posterior (M-P)) in KOA patients (n = 10) using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at 20 μm spatial resolution. The degree of cartilage degeneration was evaluated histologically, and the subchondral bone tissue microarrays (TMAs) were subsequently manually constructed from formalin-fixed paraffin-embedded (FFPE) KOA osteochondral (i.e., cartilagesubchondral bone) tissues. Overall, the Osteoarthritis Research Society International (OARSI) histological grade was significantly higher and the size of chondrocytes in the superficial zone was much larger for both M-A and M-P compared to L-A and L-P of cartilage (p = 0.006, p = 0.030, p = 0.028, and p = 0.010; respectively). Among the 65 putative N-glycans observed by MALDI-MSI, 2 core fucosylated bi-antennary N-glycans, m/z 1809.64; (Hex) 5 (HexNAc) 4 (Fuc) 1 and 2100.73; (NeuAc) 1 (Hex) 5 (HexNAc) 4 (Fuc) 1 , were significantly higher in intensity in M-A compared to L-A of the trabecular bone (p = 0.027, and p = 0.038, respectively). These N-glycans were then further structurally characterized by in situ MS/MS fragmentation post-MALDI-MSI. Our results demonstrate, for the first time, N-glycan alterations can occur at different joint loading forces in the KOA tibial plateau and the feasibility of subchondral bone TMA construction for N-glycan MALDI-MSI.

Mass Spectrometry Imaging of the Subchondral Bone in Osteoarthritis Reveals Tissue Remodeling of Extracellular Matrix Proteins that Precede Cartilage Loss

“…Briefly, glycan imaging identified 46 glycan species from a database of 165 known glycan species in FFPE sections of intact human tibial plateaus from a subset of 3 OA patients (Supplemental Table 2). Comparing the lateral and medial halves of the three imaged patients (OA patients 2,3,4), some interesting spatial patterns were observed, such as a tendency for glycan signal to change in areas of subchondral bone damage in OA tissues, but significantly regulated glycans in OA were not yet detected, though other studies that have used large cohorts leveraging tissue microarrays do discriminate glycan changes in subchondral bone [37,38]. Some glycans with preferential abundance for cartilage or bone regions were observed, but these did not appear to be disease regulated.…”

“…Given the mixed tissue types within the joint, such as mineralized bone and the highly hydrated cartilage, no commonly used tissue preparation strategy for MALDI-MSI is appropriate for studying all the different compartments of the joint and different analytes, from small molecule metabolites and lipids to larger proteoglycans, proteins, and peptides. While others have utilized MALDI-MSI to visualize changes to lipids and proteoglycans in bone [29,33,[35][36][37][38]63] protein-based imaging of joint tissues has been thus far limited to the synovium and cartilage, which even then has resulted in a small number of detected proteins. Partially, this is due to the nature of the skeletal ECM, especially in bone, which is highly crosslinked and post-translationally modified with glycans that altogether act as a physical barrier impeding the access of traditional enzymes used in MS strategies such as trypsin.…”

“…Recently, spatial profiling of the subchondral bone has been achieved for lipids, expanding MALDI-MSI to mineralized tissues [33][34][35][36]. Other investigators have utilized MALDI-MSI to study N-glycans in the subchondral bone of the knee in OA and identify specific glycans within subchondral trabecular bone that are upregulated in different joint quadrants that may correspond to areas of altered mechanical loading with disease [29,37,38]. While these studies represent advancements in the use of MALDI-MSI overall in subchondral bone, protein imaging of joint tissues has thus far been limited to the synovium and articular cartilage in human and equine samples and not yet bone tissue [29,39,40].…”