Graduate Research Assistant University of Maryland, Fischell Department of Bioengineering West Friendship, Maryland, United States
Introduction: : Uterine fibroids are the most common gynecological tumor, affecting up to 80% of women by age 50 [1]. Despite fibroid prevalence, the cellular mechanisms underlying fibroid development remain poorly understood due to the lack of physiologically relevant models for fibroids. Fibroid tissues isolated from patients provide insight into tissue architecture and biological components of fibroids but have limited capacity for mechanistic studies. Current in vitro models for fibroids do not recapitulate fibroid mechanics, 3D structure, and extracellular matrix (ECM), which limits our understanding of fibroid pathogenesis.
A hallmark of fibroids is the excessive ECM accumulation relative to the myometrium. This increased ECM contributes to elevated tissue stiffness, which influences fibroid growth and symptom progression through mechanotransduction–a process in which mechanical cues from the ECM trigger intracellular signaling pathways [2]. These cell–ECM interactions are highly dynamic, as cells both remodel their surrounding matrix and respond to its biophysical properties.
Fibroblasts play a pivotal role in fibroid development through ECM deposition and remodeling. Upon activation by transforming growth factor-beta (TGF-β), fibroblasts can differentiate into myofibroblasts, proliferate, and secrete ECM. In fibroid tissue, elevated levels of TGF-β3 are accompanied by increased proliferation, ECM accumulation, and stiffness compared to healthy myometrium [3,4].
To study fibroid development in a physiologically relevant model, we developed and optimized a poly(ethylene glycol) (PEG)-based hydrogel that incorporates fibroid cellular components, ECM-derived peptides, and bulk stiffness. We hypothesize that TGF-β3 activates uterine fibroblasts to produce ECM proteins in our PEG-based hydrogel model, recapitulating in vivo fibrotic behavior.
Materials and
Methods: : Hydrogel fabrication: Hydrogels were functionalized with the cell-adhesive peptide RGDS and the enzyme-cleavable peptide GGGPQGIWGQGK (PQ). Peptide concentrations were optimized to support cell adhesion and elongation while recapitulating the matrix stiffness of native fibroid and healthy myometrial tissues.
Cell source: Primary human uterine fibroblasts isolated from myometrial tissue were used between passage 3-6 for hydrogel encapsulations..
TGF-β3 activation: Uterine fibroblasts were encapsulated in PEG hydrogels and treated with 0 ng/mL (control), 10 ng/mL, and 20 ng/mL TGF-β3 for 7 days.
Immunocytochemistry: After 7 days, PEG hydrogels were fixed and stained for alpha smooth muscle actin (α-SMA), collagen I (Col1), Col3, proliferation marker Ki67, fibronectin (FN), phalloidin via F-actin, and cell nucleus (DAPI). Confocal imaging was then performed to visualize ECM proteins and cell morphology, normalizing to the number of DAPI counts.
ECM/Morphology Analysis: Cell counts were quantified based on the DAPI channel using IMARIS spot analysis. Total volume of aSMA and Col1 production was quantified using IMARIS surface analysis and normalized to the number of DAPI counts. Cell elongation was inferred from the cell-to-segment ratio, using the DAPI and phalloidin channels.
Statistical Analysis: Using GraphPad Prism, experiments were analyzed using one-way ANOVA followed by Tukey’s post hoc tests.
Results, Conclusions, and Discussions:: To develop a physiologically relevant platform for studying uterine fibroblast activation, we first optimized the hydrogel formulation by tuning the concentration of cell-adhesive peptide PEG-RGDS. Human uterine fibroblasts were encapsulated in 6% PEG-PQ-PEG hydrogels with either 3.5 mM or 2 mM PEG-RGDS and cultured for 7 days with untreated, 10 ng/mL, or 20 ng/mL TGF-β3. At 2 mM PEG-RGDS promoted a more elongated fibroblast morphology upon TGF-β3 stimulation compared to the other conditions (Fig. 1B). These results demonstrate that 2 mM PEG-RGDS more effectively supports fibroblast elongation.
To mimic the bulk mechanical stiffness of myometrial tissue, we encapsulated uterine fibroblasts in 4% or 6% PEG-PQ-PEG hydrogels, modeling the stiffness of myometrium and fibroid tissues, respectively. All conditions used 2 mM PEG-RGDS. Following 7 days of encapsulation with or without TGF-β3 treatment, we assessed fibroblast activation via expression of α-SMA and Col1. Confocal imaging revealed elevated α-SMA and Col1 volume in TGF-β3-treated samples across both stiffness conditions (Fig. 1C & 1D). For the 4% PEG-PQ-PEG hydrogels, 20 ng/mL TGF-β3 increased α-SMA compared to no treatment (p < 0.05) . For the 6%, both 10 ng/mL and 20 ng/mL significantly increased α-SMA and Col1 compared to no treatment (p < 0.01). We next examined the relationship between cell proliferation and ECM production. At 24 hours post-encapsulation, fibroblasts exhibited high proliferative activity. However, by days 4 and 6, a phenotypic shift was observed, with cells transitioning toward a matrix-producing phenotype characterized by increased deposition of collagens. Additionally, fibroblast elongation was inferred from the cell-to-segment ratio, with higher values indicating more elongated or connected cells. TGF-β3 had no effect in 4% hydrogels but significantly increased elongation in 6% hydrogels compared to untreated samples (p < 0.01) (Fig. 1E).
Together, these findings establish that PEG-based hydrogels with tunable stiffness and optimized cell-adhesion support TGF-β3-mediated activation of uterine fibroblasts. This system recapitulates the key biochemical and mechanical cues of both healthy and fibroid-like uterine environments, making it a valuable tool for modeling fibrotic remodeling in fibroid pathophysiology.
