Objective: During osteoarthritis (OA), chondrocytes seem to change their spatial arrangement from single to double strings, small and big clusters. Since the pericellular matrix (PCM) appears to degrade alongside this reorganisation, it has been suggested that spatial patterns act as an image-based biomarker for OA. The aim of this study was to establish the functional relevance of spatial organisation in articular cartilage. Method: Cartilage samples were selected according to their predominant spatial cellular pattern. Young's modulus of their PCM was measured by atomic force microscopy (AFM) (~500 measurements/pattern). The distribution of two major PCM components (collagen type VI and perlecan) was analysed by immunohistochemistry (8 patients) and protein content quantified by enzyme-linked immunosorbent assay (ELISA) (58 patients). Results: PCM stiffness significantly decreased with the development from single to double strings (p ¼ 0.030), from double strings to small clusters (p ¼ 0.015), and from small clusters to big clusters (p < 0.001). At the same time, the initially compact collagen type VI and perlecan staining progressively weakened and was less focalised. The earliest point with a significant reduction in protein content as shown by ELISA was the transition from single strings to small clusters for collagen type VI (p ¼ 0.016) and from double strings to small clusters for perlecan (p ¼ 0.008), with the lowest amounts for both proteins seen in big clusters. Conclusions: This study demonstrates the functional relevance of spatial chondrocyte organisation as an image-based biomarker. At the transition from single to double strings PCM stiffness decreases, followed by protein degradation from double strings to small clusters.

During osteoarthritis, chondrocytes change their spatial arrangement from single to double strings, then to small and big clusters. This change in pattern has recently been established as an image‐based biomarker for osteoarthritis. The pericellular matrix (PCM) appears to degrade together alongside cellular reorganization. The aim of this study was to characterize this PCM‐degradation based on different cellular patterns. We additionally wanted to identify the earliest time point of PCM‐breakdown in this physiopathological model. To this end, cartilage samples were selected according to their predominant cellular pattern. Qualitative analysis of PCM degradation was performed immunohistochemically by analysing five main PCM components: collagen type VI, perlecan, collagen type III, biglycan, and fibrillin‐1 (n = 6 patients). Their protein content was quantified by enzyme‐linked immunosorbent assay (127 patients). Accompanying spatial cellular rearrangement, the PCM is progressively destroyed, with a pericellular signal loss in fluorescence microscopy for collagen type VI, perlecan, and biglycan. This loss in protein signal is accompanied by a reduction in total protein content from single strings to big clusters (P < .001 for collagen type VI, P = .003 for perlecan, and P < .001 for biglycan). As a result of an increase in the number of cells from single strings to big clusters, the amount of protein available per cell also decreases for collagen type III and fibrillin‐1, where total protein levels remain constant. Biochemical changes of the PCM and cellular rearrangement are thus highly interconnected hallmarks of osteoarthritis. Interestingly, the earliest point in time for a relevant PCM impairment appears to be at the transition to small clusters.

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