Prevention of Muscle Fibrosis in Duchenne Muscular Dystrophy via Engineered Mesenchymal Stromal Cell-Mediated Fibronectin Degradation

Abstract

Duchenne Muscular Dystrophy (DMD) is a genetic disorder caused by the loss of the proteindystrophin, resulting in progressive muscle degeneration and impaired regeneration. A hallmark feature of DMD pathology is excessive extracellular matrix (ECM) deposition, marked by early fibronectin accumulation followed by collagen-rich fibrosis. Currently, no therapies effectively prevent or reverse fibrosis in DMD, underscoring the need for new regenerative approaches. Mesenchymal stromal cells (MSCs) have emerged as a promising cell-based therapy due to their ability to secrete a broad array of paracrine factors, including matrix-remodeling enzymes. Despite evaluation in over a thousand clinical trials, MSC-based therapies have yielded inconsis tent outcomes, in part due to limited control over MSC behavior following administration. Given the mechanosensitivity of MSCs, which enables them to adapt functionally to their physical mi croenvironment, we sought to develop a mechanobiological strategy to enhance the therapeutic potential of MSC-derived secretomes for muscle fibrosis. Wehypothesized that conditioning MSCs within tunable hydrogel environments would enrich their secretion of matrix metalloproteinases (MMPs), thereby promoting fibrotic ECM remodeling in DMD. D1 murine MSCs were cultured on tissue culture plastic or encapsulated within alginate hydrogels of defined stiffness (~2.9–5.0 kPa) and stimulated with tumor necrosis factor–α (TNFα). Expression and secretion of MMP 2, 3, 9, and 10 were quantified using qPCR and ELISA. MSCs encapsulated in softer hydrogels exhibited enhanced MMP expression, with MMP-3 showing the most pronounced transcriptional upregulation, which was subsequently confirmed at the protein level. Conditioned media from engineered MSCs was then administered via intramuscular injection into wild-type and dystrophin-deficient mice to assess therapeutic efficacy. Treated dystrophin deficient mice demonstrated improved locomotor activity, alongside reduced fibronectin and col lagen deposition within muscle tissue, as assessed by immunohistochemistry. Collectively, these findings demonstrate that hydrogel-mediated mechanobiological condition ing enhances the ECM-remodeling capacity of engineered MSCs, leading to reduced fibrotic de position and improved muscle function in vivo. This work supports the development of a novel therapeutic approach for managing muscle fibrosis in Duchenne Muscular Dystrophy.