A Biomaterial Model of Uterine Fibroids: Understanding Fibroblast Differentiation into Myofibroblasts

Introduction: Uterine fibroids are the most common gynecological tumor, affecting 70 - 80% of women aged 35 - 49 [1]. However, the cellular mechanisms behind fibroid development remain severely understudied due to the lack of relevant research models for fibroids. Current research methods rely on whole-patient tissue analysis, which have provided valuable findings regarding the components of the fibroid microenvironment but are extremely difficult and invasive to attain.

We know that macrophages are higher in density in fibroids compared to distant myometrial tissue [2], which suggests a potential immunomodulatory role of macrophages in fibroid development. In addition to increased macrophages, transforming growth factor beta 3 (TGF-β3) and the extracellular matrix (ECM) deposition are increased in fibroid tissue [3,4] Interestingly, in other conditions involving fibrous accumulation, pro-fibrotic macrophages are well-known to secrete TGF-β to activate fibroblasts to produce excessive ECM. However, neither whole fibroid tissue analysis nor any relevant fibroid research model allow insight into the mechanistic role of macrophages in fibroid development.

To unravel macrophage modulation on fibrotic pathways in fibroid development, we must design a physiologically relevant platform that mimics the fibroid microenvironment. To address this current gap in knowledge, we developed a tissue-engineered poly(ethylene glycol) (PEG)-based hydrogel model to investigate human uterine fibroblast activation upon TGF-β3 treatment. We hypothesized that macrophage-secreted TGF-β3 activates uterine fibroblasts to differentiate into myofibroblasts and produce ECM in our PEG-based hydrogel.

Materials and

Methods: PEG hydrogel fabrication: PEG hydrogels were functionalized with a cell adhesive peptide RGDS and enzyme-cleavable peptide GGGPQGIWGQGK (PQ). Combining 6 w/v% PEG-PQ-PEG and 3.5 mM PEG-RGDS to simulate the matrix stiffness of native fibroid tissue.

Cell source: Primary human uterine fibroblasts isolated from myometrial tissue were used between passage 3 - 5 for hydrogel encapsulations. Uterine fibroblasts were cultured for 5 days prior to encapsulation.

Cell viability: Uterine fibroblasts were encapsulated in the hydrogel for 7 days and assessed for viability and proliferation using the LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher). Live and dead cells were quantified using Analyze Particles in ImageJ.

TGF-β3 activation in PEG hydrogel: Uterine fibroblasts were encapsulated in PEG hydrogel for 5 days and treated with 0 ng/mL (control), 0.1 ng/mL, 1 ng/mL, and 10 ng/mL TGF-β3 concentrations for 48 hours.

Immunocytochemistry: After 7 days, PEG hydrogels were fixed and stained for alpha smooth muscle actin (α-SMA), collagen I (COL1A1), and cell nucleus (DAPI) to determine fibroblast ECM production and cellularity. Immunofluorescence confocal imaging was then performed to visualize and quantify ECM proteins and uterine fibroblasts.

Results, Conclusions, and Discussions: After 7 days of encapsulation, fibroblast viability was assessed (Fig. 1A). Comparing confocal microscopy images, live and dead cells were quantified. Greater than 90% of the cells remained alive. This result indicates that the PEG-based hydrogel can support uterine fibroblast cell viability over 7 days.

Because the fibroblasts showed that they remained viable for 7 days in the hydrogel, we encapsulated uterine fibroblasts in hydrogel for 5 days and then investigated fibroblast cell response to 0 ng/mL, 1 ng/mL, 10 ng/mL, and 100 ng/mL TGF-β3 treatments for the remaining 48 hours. Hydrogels were analyzed for fibroblast activation, or myofibroblast phenotype. We were particularly interested in ECM components that signify myofibroblasts, including α-SMA and collagen I (see Figure 1B for workflow). Using confocal microscopy, we quantified these components and compared across treatment groups. Upon increasing TGF-β3 treatments, increased α-SMA and COL1A1 levels were observed. Based on increased ECM production, encapsulated uterine fibroblasts exposed to TGF-β3 resemble the activation response of fibroblasts in native fibroids.

M2 macrophages have been shown to secrete increased TGF-β3 in several types of fibrosis. Uterine fibroblast activation in response to TGF-β3 treatments in our hydrogel model suggests that the high levels of TGF-β3 in fibroids may be secreted by M2 macrophages. This key finding will inform future fibroid research including macrophage and uterine fibroblast co-culture studies to investigate direct cell-cell interactions in the fibroid-like microenvironment.

In this study, uterine fibroblast activation by TGF-β3 establishes our PEG hydrogel system as capable of mimicking the fibroid microenvironment. Thus, our hydrogel system exhibits potential as a proof-of-concept model for the activation of fibroblasts during uterine fibroid development. In our future investigations, we may use this model to further investigate the specific mechanisms governing fibroid development via macrophage and fibroid cell co-culture. This in vitro model will allow future studies involving fibroid cell-cell interactions to reveal key insights into fibroid pathogenesis. Once we determine the mechanisms mediating fibroid growth, we will use this model to identify key targets for treating or preventing uterine fibroids to eliminate the burden faced by millions of women.

Acknowledgements (Optional):