Human Tendon-on-a-Chip for modeling vascular inflammatory fibrosis
Vascular inflammation and activation of myofibroblasts play crucial roles in the progression of fibrosis. Transforming growth factor beta 1 (TGF-ß1) has been identified as a driver of adhesion formation in various tissues, including tendons. However, the mechanisms underlying fibrotic peritendinous adhesions remain poorly understood resulting in a lack of effective therapies. To address this, we developed a novel human Tendon-on-a-Chip (hToC) model, which combines a vascular compartment, with endothelial cells and monocytes, with a tissue compartment containing fibroblasts and tissue-resident macrophages, all in serum-free conditions. Our hToC successfully replicates inflammatory and fibrotic phenotypes observed in mouse models and clinical human samples including myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and secretion of inflammatory cytokines. We show that fibrosis-on-a-chip is driven by the interaction between the vascular and tissue compartments, including the infiltration of monocytes. Transcriptomics validate the hToC as disease model of diseased human tendon and shows the upregulation of the PI3K/AKT/mTOR pathway—a regulatory nexus of fibrosis in tendon injury. Consistent with this finding, treatment with the mTOR inhibitor Rapamycin suppresses the fibrotic phenotype. Our findings validate the hToC as a tool for investigating human fibrosis and illuminate the underappreciated vascular contribution to tendon pathophysiology. Overall design: We developed an in vitro human tendon-on-a-chip (hToC) platform quad culture model composed of human primary tenocytes (TCs) and tissue macrophages (tM?) in a collagen hydrogel model of injured tendon, combined with human umbilical endothelial cells (ECs) and circulating Monocytes (cM?) to model the new vasculature in injured tendon. To introduce patient variability, we used three distinct M? as donor 1, donor 2, and donor 3. All the other cell sources (TCs and ECs) were kept consistent (primary TCs derived from a single patient of hand surgery, and HUVECs were purchased from a commercial source). Since mTOR was a consistent signature of inflammation and fibrosis in the hToC upon the introduction of TGF-ß1, we treated the disease devices (TGF-ß1 treated hToC) with Rapamycin, an mTOR inhibitor, and compared them with TGF-ß1 treated diseased hToC devices ( injured controls). We performed bulk RNA-sequencing of the two-compartment hToC to evaluate the effects of Rapamycin.