Introduction: Methods: Synovial biopsies from seven patients were exposed to three inoculate doses of Bb (250k, 500k, 1000k) in our Joint Space Analysis System (JSAS). Tissue samples and supernatant were collected at 24, 48, and 72 hours for histology, immunofluorescence (IF), RNAscope, and ELISA. Results: We found dose- and time-dependent changes in synovial intimal lining (IntL) thickness and cellularity. IntL integrity significantly decreased with 1000k Bb at Day 2 (p = 0.021) and remained decreased with 1000k Bb at Day 3 (p = 0.005). IF revealed sustained expression of the M2 marker CD206 in joint-lining macrophages despite infection. RNAscope demonstrated a spatially limited co-localization of Bb within the tissue and alteration of tight junction ZO-1 subcellular localization within the IntL. ELISA indicated reduced Osteoprotegerin at Day 3, suggesting potential for altered bone remodeling activity. IL-10 secretion increased over time and Bb dose. Discussion: These findings demonstrate that Bb interaction with joint-lining macrophages is associated with rapid, structural, and functional alterations, with loss of IntL integrity and macrophage dysregulation. Compromise of the synovial barrier may facilitate immune cell infiltration, contributing to disease pathogenesis, in the setting of increased markers of synovitis and increased regulatory cytokines, but causative association of these phenomena remains to be explored.