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Tissue Engineering

Tissue Engineering - Poster Session D

Poster K17 - Modulation of fibrosis in 3D Collagen Gel Model: Impact of Col VI and CCN1

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The cardiac extracellular matrix plays a critical role in maintaining the structural strength of the myocardium, its composition can be significantly altered by an acute injury such as myocardial infarction. Cardiac fibrosis, caused by the excessive production of extracellular matrix, facilitates progression of arrhythmia and cardiac dysfunction. People who develop cardiac fibrosis have an increased risk for heart failure and death. Currently there are not feasible therapies to reverse the effects of chronic fibrosis. Our goal is to establish and evaluate an effective 3D model of fibrosis to investigate the mechanisms of tissue repair providing a significant move toward creating more effective regenerative therapies for patients suffering with heart disease. The objective of this study is to evaluate a 3D collagen model of cardiac fibrosis that investigates the impacts of Collagen VI (Col VI) and CCN1 I (matricellular protein also known as CYR61; cysteine-rich protein 61) on fibroblast proliferation and function. Both, CCN1 and Col VI, can influence fibrotic depositions, making them significant proteins to be incorporated in 3D engineered fibrosis models.

Materials and

Methods:  The model consists of fibroblasts seeded within a 3D collagen type 1 gel (2.5mg/mL). To identify changes in fibroblast function, the models are also prepared with or without incorporation of CCN1 or Col VI. Cells are seeded within these gels at a density of 4000 cells per cm2 and cultured for five days in a 96-well plate. Samples are analyzed via fluorescent imaging analysis, MTT analysis, and gene expression is analyzed via qPCR. Cells are stained using Alexa Fluor 568-phalloidin to stain for F-actin, immunostaining to stain for alpha-smooth muscle actin (α-SMA), and nuclei are stained with DAPI. We analyze changes in fibroblast phenotype, proliferation, and α-SMA expression using fluorescence imaging and Image J. Quantitative polymerase chain reaction (qPCR) using SYBR Green is also performed for the following targets: GAPDH, α-SMA, Cyclin D1. Samples were also analyzed using an MTT assay to correlate and compare cell number between samples.

Results, Conclusions, and Discussions: Our results demonstrate that fibroblasts cultured in our CCN1 model showed a decrease in proliferation compared to the collagen model without CCN1. Additionally, there was a trend of decreased expression of α-smooth actin in samples with CCN1. The data also indicates that fibroblasts cultured in our collagen type VI model had increased proliferation and increased expression of α-SMA. Imaging of samples also showed differences in cell shape and size in the different models. 

Overall, our studies demonstrated that we can develop models of fibrosis that modulate fibroblast function, the presence of collagen type VI, which is known to be a key ECM component of post-MI remodeling, can induce fibroblast activation and myofibroblast differentiation, whereas incorporation of CCN1 results in a more quiescent fibroblast phenotype and function during wound healing. Current work is also focused on analyzing models where multiple components are included simultaneously (for example, both ColVI and CCN1). 

We have developed physiologically relevant 3D models of fibrosis that can modulate fibroblast function to increase activation and proliferation, and models that can decrease fibroblast activation and proliferation. These models can help identify potential therapeutic targets, and they could also be used as early pre-clinical testing models for translational studies. These results will contribute to the development of novel approaches to modulate the balance between pathological fibrosis and endogenous mechanisms of regeneration and repair.

Acknowledgements (Optional):  Acknowledgements: This research was supported by the National Science Foundation CAREER Award, as well as the NSF REU at CSULB.