Single-nucleus multiome profiling reveals osteocyte-centered remodeling of human subchondral bone across osteoarthritis severity

Objective: Subchondral bone remodeling is a hallmark of osteoarthritis (OA), but osteocytes, the principal regulators of bone homeostasis, have been inaccessible to single-cell studies because they are embedded in mineralized matrix. We aimed to define the cellular, transcriptional and chromatin changes of human subchondral bone across OA severity, osteocytes included, and to identify candidate signals between cartilage and bone. Design: Subchondral bone from the medial femoral condyle of human knees graded by the Outerbridge system was analyzed by bulk RNA sequencing (89 knees) and by single-nucleus multiome sequencing of gene expression and chromatin accessibility after mechanical nuclear isolation (16 donors). Three hypotheses were prespecified: with increasing OA severity, (1) the osteoprogenitor fraction increases, (2) the osteocyte fraction increases, and (3) osteocyte SMAD2/3 motif activity increases. Results: With OA severity, bulk expression shifted toward extracellular matrix and ossification programs. The atlas resolved 26 cell states among 61,570 nuclei, including 906 osteocytes. Osteoprogenitors (p = 0.003) and osteocytes (p = 0.036) increased in share along an osteogenic continuum that starts from CXCL12-abundant reticular cells. Chondrocytes at the osteochondral junction upregulated FN1, FGF1, FGF2 and TNFSF11 without increased hypertrophic gene expression, and their share of the inferred FN1 signal received by osteocytes rose from 4% in normal knees to 33% in severe OA. Osteocyte SMAD2/3 motif activity increased with severity (rho = 0.70, p = 0.003), and all three hypotheses remained significant after Holm correction. Across the osteogenic lineage, SMAD2/3 activity correlated with junction chondrocyte FN1 (rho = 0.63, p = 0.017), and in osteocytes it was accompanied by TGF-β-responsive matrix genes and perilacunar-remodeling genes. Conclusions: This osteocyte-inclusive atlas of human OA subchondral bone shows expansion of the osteogenic compartment and a rise of osteocyte SMAD2/3-linked matrix remodeling with disease, and identifies chondrocyte-derived FN1 as a candidate contributor to cartilage-bone communication associated with osteocyte TGF-β/SMAD2/3 signaling.