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Introduction Calcinosis is a debilitating manifestation of systemic sclerosis (SSc), affecting up to 20% of patients, and is associated with long disease duration, digital ulcers, and acro-osteolysis. It results from ectopic deposition of hydroxyapatite in skin and subcutaneous tissue, causing pain, infections, and functional impairment. The pathogenesis remains poorly understood due to lack of suitable models. Current evidence suggests mesenchymal stem cells (MSCs) undergo osteogenic differentiation under inflammatory and mechanical stress, while macrophages contribute to fibrosis and calcinosis by secreting osteogenic mediators such as TGF-β, BMP-2, and Activin A.Material and Methods We established a co-culture model of adipose-derived MSCs differentiated towards osteocytes with peripheral blood–derived macrophages from diffuse cutaneous SSc patients and healthy controls. Cultures were maintained in osteogenic medium, with macrophages introduced at day 14 and 17 of differentiation. Calcium deposition was assessed by Alizarin Red S staining at day 21, and Activin A release was measured in plasma and culture supernatants by ELISA (Biotechne, Quantikine Activin A, Cat. #353043). Pharmacological inhibition with a CD206 antagonist(10 µM) and an Activin A neutralising antibody (RCD Systems, 10 µg/mL) was used to assess therapeutic potential. Complementary immunohistochemistry was performed on calcinotic skin biopsies to validate in vitro findings.Results Plasma Activin A levels were significantly elevated in SSc compared with healthy controls, particularly in the anti-centromere antibody subgroup (521 ± 199 vs 255 ± 170 pg/mL, p<0.001). SSc-derived macrophages secreted enhanced osteogenic foci in MSC co-cultures compared with controls (1.33 ± 0.29 vs 0.83 ± 0.29, p<0.05). Alizarin Red staining confirmed increased hydroxyapatite deposition, which was attenuated by CD206 inhibition (0.33 ± 0.29, p<0.01) and Activin A neutralisation (0.50 ± 0.29, p<0.05). CD206 inhibitor treatment significantly modulated macrophage activation marker expression (CD206 by qPCR p=0.0006).Conclusions This study demonstrates that SSc macrophages promote calcinosis by driving MSC osteogenic differentiation and implicates the possible role of Activin A release and CD206-dependent pathways. The novel co-culture model provides a reproducible platform for mechanistic studies and therapeutic testing. Ongoing work with excised calcinosis samples, analysed by immunochemistry, will further link macrophage– stromal cross-talk to in situ calcinosis. Collectively, these efforts highlight macrophage-driven signalling to MSCs as a promising therapeutic target for this severe and treatment-resistant complication of SSc.Abstract P.044 Figure 1