Pirfenidone Attenuates Fibrosis and Neovascularization in 3D Spheroid-Laden Hydrogel Culture

Fibrosis and angiogenesis are key contributors to synovial inflammation in both the early and progressive stages of rheumatoid arthritis (RA) and osteoarthritis (OA), making them important therapeutic targets to mitigate joint tissue damage. In vitro drug screening, particularly for antifibrotic and antiangiogenic efficacy, is a standard method for evaluating therapeutic candidates prior to in vivo testing. Traditionally, most studies have relied on two-dimensional (2D) monolayer cell cultures, which lack physiologically relevant cell-matrix and cell-cell interactions. Substantial evidence now indicates that three-dimensional (3D) culture systems more accurately recapitulate the structural and functional complexity of native tissue environments. We employed 3D spheroid culture models of fibrosis and neovascularization to evaluate the antiangiogenic and antifibrotic effects of pirfenidone (PFD), an FDA-approved drug for idiopathic pulmonary fibrosis. Spheroid monocultures of 3T3 fibroblasts and co-cultures of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (SMCs) were encapsulated in cell-adhesive, proteolytically degradable polyethylene glycol (PEG) hydrogel scaffolds. The temporal effects of PFD dose and timing of addition in culture on fibroblast outgrowth, vascular sprouting, and viability were quantified up to 14 days. PFD treatment led to dose-dependent inhibition of both fibroblast outgrowth and vascular sprouting, depending on the initial timing of PFD addition, with cell viability maintained under all conditions. In addition, PFD reversed the onset of fibrosis and neovascularization. PFD exhibited antifibrotic activity and antiangiogenic potential in 3D cultures.