MAKING WAVES: ULTRASOUND-STIMULATED ORGANOIDS TO MODEL FROZEN SHOULDER

Frozen shoulder (FS) resolution involves key roles for macrophages and fibroblasts (Ng et al., 2023). We developed a 3D patient-derived organoid (PDO) model to improve understanding of fibrosis resolution and identify potential anti-fibrotic drug targets. Primary fibroblasts from FS and comparator tissue were combined with primary macrophages, HUVECs, and Matrigel™ to create PDOs. PDOs were sectioned and stained with Ki67, CD146, to visualize fibroblast activation and proliferation. Markers DKK3, CD31 and POPO1 were used to view fibroblasts, HUVECs and nuclei respectively. MerTK expression in macrophages was induced in 2D using LPS, DEX, PROS1, and ADAM17 inhibitor, followed by FACS analysis. To investigate mechanical stimulation, fibroblast-containing organoids were subjected to ultrasound treatment and subsequently sectioned and stained for mechanosensitive markers TRPV4, IL-R1, vinculin, and Piezo1. FS PDOs displayed a thickened lining layer, elevated CD146, and increased Ki67 expression, reflecting fibroblast activation and proliferation. Z-stack imaging revealed evenly distributed HUVECs forming tube-like network in PDOs. 3D projections showed a more defined HUVEC network in FS PDOs, reflecting the disease's heightened vascularization. DEX induced the highest MerTK expression, followed by PROS1 and unstimulated cells, while LPS reduced it. ADAM17 inhibition decreased MerTK shedding. Mechanical stimulation enhanced cell viability and upregulated IL-R1 and vinculin, aligning with FS tissue characteristics. This PDO model recapitulates key FS tissue features such as thickened capsule lining and increased vascularity. Without representative animal models, this PDO platform offers a novel tool for studying disease mechanisms and testing therapies. We successfully identified and refined two highly desirable features for our PDO model: methods to polarize macrophages toward pro-resolving or pro-inflammatory phenotypes in 2D, and ultrasound-mediated mechanical stimulation. A: H&E stained sections of patient synovial tissues taken from a comparator or frozen shoulder. B: D10 organoids stained with CD146 (red), Ki67 (yellow), and POPO-1 (cyan). Arrows indicate thickened lining layer. A: whole organoid imaging demonstrating distribution of HUVECs (CD31, green), and fibroblasts (DKK3, red), with nuclear stain POPO-1 (blue). B: 3D projections of PDOs reveal increased HUVEC tube formation in frozen shoulder PDO (arrows).