Poster BB2 - Development of 3D Fibrosis Model: Effect of TGF-β1 and Matrix Density on Fibrosis Progression

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

JM

Jacqueline Mejia

Research Lab Assistant
California State University Long Beach
Santa Ana, California, United States

Primary Investigator(s)

Perla Ayala, PhD

Associate Professor
California State University Long Beach, California, United States

Presenting Author(s)

Valeria Garcia (she/her/hers)

Researcher
CSULB
Cypress, California, United States

Introduction: Cell death after myocardial infarction leads to an uncontrolled reparative response, where fibroblast cells become activated to produce chronic fibrotic scar tissue. Cardiac fibrosis is characterized by excessive collagen and extracellular matrix (ECM) deposition that disrupts cardiac function and can lead to heart failure. In vitro 3D models able to mimic the complex mechanisms involved in cardiac fibrosis are crucial for the development of antifibrotic therapies and developmental studies. This study is focused on developing and validating our 3D fibrosis model by investigating the effects of TGF-β1 and matrix density within this system.

Materials and

Methods: Our current model consists of a 3D collagen type I hydrogel modified to evaluate fibroblast function and fibrosis progression. For this study, 3T3 fibroblasts were cultured within the 3D collagen type I hydrogel model for 5 days. To investigate the effect of matrix density, hydrogels were prepared at two different densities (2.5 mg/ml and 4.0 mg/ml). One set of models was developed using rat tail tendon collagen and another set of models was developed using porcine skin derived collagen. To investigate the role of biochemical factors on fibrosis progression in our models, media was supplemented with TGF-β1 for the experimental samples. Fibroblast proliferation and activation was assessed after culturing the samples for five days. The samples were stained to identify the nuclei (with DAPI), F-actin (Alexa Fluor 568-phalloidin), and immune-stained for alpha-smooth muscle actin (⍺-SMA). Image analysis was done to quantify cell counts and fluorescence intensity using image J. Quantitative Polymerase Chain Reaction (qPCR) was performed to analyze gene expression of ⍺-SMA, cyclin D1, and collagen type I.

Results, Conclusions, and Discussions: Our results indicate that our 3D models exhibit changes in fibroblast proliferation and fibrosis progression based on matrix density and the incorporation of biochemical factors such as TGF-β1. Rat tail tendon collagen samples at 2.5 mg/ml showed a significant increase in α-SMA and cyclin D1, and an increasing trend in collagen type I when cultured with TGF-β1, as assessed by qPCR and immunostaining. Having access to a higher density with the rat tail tendon collagen presented challenges, due to commercial availability of higher collagen concentrations, whereas porcine skin collagen was extracted in house allowing us further control over collagen density.

In porcine skin-derived collagen samples fibroblast proliferation was significantly higher in the 4.0 mg/ml samples compared to the lower density samples. Gene expression analysis results for these samples also supported these findings, with a significant increase in α-SMA and collagen type I expression in the 4.0 mg/ml samples compared to the 2.5 mg/ml samples. Additionally, the 4.0 mg/ml TGF-β1 treated samples exhibited higher α-SMA expression compared to the control samples. The 2.5 mg/ml samples cultured with TGF-β1 demonstrated a significant increase in collagen type I expression.

This study demonstrated that TGF-β1 influences fibroblast activation and proliferation in 3D collagen models, with matrix density playing a crucial role. Future work will focus on MTT assays to further assess cell proliferation. These results align with established knowledge of fibrosis progression and highlight the significant role 3D models of fibrosis can provide for investigating therapeutic candidates for the modulation of cardiac fibrosis to prevent or reverse cardiac fibrosis and heart failure.

Acknowledgements (Optional): This research was supported by the National Science Foundation CAREER Award, as well as funding support from the Learning Aligned Employment Program and the NSF REU at CSULB.