An in vitro model to measure the strength and stiffness of the extracellular matrix synthesized de novo by human fibroblasts.

Abstract

Purpose: Alterations to the strength and stiffness of the human extracellular matrix (ECM) are the underlying pathology manifest in a wide range of diseases. These include inherited conditions, such as Ehlers Danlos syndrome, as well as acquired diseases such as fibrosis, which remains a major unmet medical need. To evaluate promising therapies, new models are needed that can measure the strength and stiffness of the human ECM.

Methods: Cultured human fibroblasts were seeded into circular troughs of agarose that had been molded into a 24 well plate and equilibrated with cell culture medium. The cells settled by gravity, aggregated and formed 3D ring-shaped tissues 5 mm in diameter without the aid of added exogenous scaffold material. The ECM proteins synthesized de novo by the rings were characterized by immuno-staining. The response of the rings to drug and growth factor treatments were assessed by measuring changes to the dimensions of the rings and by measuring levels of collagen. A tensile test was used to quantify drug and growth factor induced changes to the strength and stiffness of the rings.

Results: Ring-shaped tissues readily formed in the molds and synthesized de novo a circumferentially aligned collagen-rich fibrous ECM network positive for collagen type I, collagen type III and fibronectin. Low dose treatment with incyclinide, an inhibitor of matrix metalloproteinases (MMPs), increased strength and stiffness, whereas as a high dose decreased tensile properties, likely due to a toxic effect. Treatment with TGF-β1, a well-known driver of fibrosis, increased levels of collagen and tensile properties and mimicked the fibrotic environment in vitro. Treatment with PAT-1251, an inhibitor of the collagen crosslinking enzyme lysyl oxidase-like protein 2 (LOXL2), had no effect on levels of collagen but significantly reduced the strength and stiffness of the ring even when elevated by treatment with TGF-β1.

Conclusion: Human fibroblasts will self-assemble a 3D ring-shaped tissue and synthesize a fibrous network of ECM proteins whose tensile properties can be measured. The fibrotic environment can be mimicked by addition of TGF-β1, which increases levels of collagen as well as the strength and stiffness of the rings. Treatment with two drugs, incyclinide and PAT 1251 that were developed as potential treatments for diseases of the ECM, altered the strength and stiffness of the rings, thereby demonstrating the utility of the model for testing new therapies that target the biomechanics of the ECM.

Supplementary information: The online version contains supplementary material available at 10.1007/s44164-025-00081-y.