BPS2026 – Scaffold-dependent matrix remodeling function of dermal fibroblasts

Fibroblasts are central regulators of tissue homeostasis, shaping extracellular matrix (ECM) architecture through deposition, alignment, and proteolysis. In turn, ECM mechanics and architecture regulate fibroblast phenotype, creating a tightly balanced reciprocity that maintains tissue integrity. Disruption of this equilibrium can drive fibrosis, chronic wounds, or tumor progression. Yet most in vitro studies rely on two-dimensional (2D) culture or 3D hydrogels with little consideration for how scaffold architecture may bias phenotype over time. To address this, we developed a pipeline in which primary human dermal fibroblasts are isolated directly from patient skin and immediately cultured in three environments: fibrous collagen hydrogels, nanoporous PEG–RGD hydrogels, and 2D tissue culture plastic (TCPS). This approach avoids extensive expansion on stiff plastic and, to our knowledge, has not been systematically applied before. We quantified proliferation, morphology, ECM deposition (collagen, fibronectin), integrin gene expression, contractility using a custom pillar-based collagen gel assay that reports both pillar deflection (cell contractility) and bulk gel compaction (matrix remodeling), and matrix metalloproteinase activity across passages, benchmarking results to native dermis using single-cell RNA sequencing datasets. Fibroblasts in both collagen and PEG hydrogels maintained balanced proliferation, physiological contractility, and stable integrin expression, consistent with physiological ECM remodeling. By contrast, fibroblasts on TCPS displayed a hyperactivated state with elevated ECM deposition, heightened baseline contractility, and altered integrin usage. Mechanistically, fibroblast contraction was TGFβ-dependent, while bulk gel compaction occurred independently of TGFβ. These scaffold-dependent differences amplified over passages, consistent with mechanical memory influencing long-term fibroblast fate. Our findings underscore the importance of scaffold architecture in shaping fibroblast function and provide direct evidence that TCPS promotes a maladaptive, non-physiological state. This work establishes a framework for selecting culture systems that preserve native fibroblast phenotype, advancing mechanobiology and translational approaches to fibrosis, wound healing, and tissue engineering.