Guiding Cell Perception of its Microenvironment for Enhanced Microfracture Repair Restricted; Files Only
Hasson, Maddie (Spring 2024)
Abstract
Introduction: Cartilage injuries impose significant societal and economic burdens, but limited treatment options are available. Microfracture is the current gold standard for cartilage injury but often leads to suboptimal outcomes, necessitating better chondrogenic therapies. The microfracture repair environment may be thoroughly explored by developing a fibrin-based gel model. Leveraging inhibition of the Rho/ROCK pathway, a signaling pathway involved in cell contractility, along with Transforming Growth Factor Beta 3 (TGF-β3), this research aims to enhance cartilage regeneration post-microfracture by elucidating cellular behavior and matrix organization within the clot environment.
Materials/Methods: Fibrin gels were synthesized and characterized by evaluating bulk mechanics, contraction, and visualizing fiber formation using scanning electron microscopy (SEM). Cell behavior in response to Rho/ROCK inhibition (Fasudil) and TGF-β3 was evaluated by quantifying cell mechanics and staining for SMAD 2/3 and stress fibers after three days. Long-term macroscale gel activity after Fasudil and TGF-β3 treatment was assessed by tracking contraction, evaluating proteoglycan deposition, and quantifying relative gene expression after four weeks in culture.
Results: After four weeks, low-thrombin fibrin gels contracted significantly compared to high-thrombin gels. Nanoindentation revealed reduced effective Young’s modulus in Rho/ROCK-inhibited cells, which was maintained after addition of TGF-β3. Stress fiber formation in Rho/ROCK-inhibited cells was attenuated compared to controls. We observed a stepwise trend in SMAD 2/3 nuclear intensity in cells treated with Fasudil, TGF-β3, and Fasudil with TGF-β3. Fasudil mitigated gel contraction compared to controls, and a 9-fold increase in type-II collagen expression was observed in gels cultured with Fasudil alone, whereas a ~200-fold increase was observed in gels cultured with Fasudil and TGF-β3.
Discussion: Higher thrombin concentrations and treatment with Fasudil modify the extracellular microenvironment and cellular machinery in a way that prevents macroscale contraction. Fasudil and TGF-β3 treatment significantly enhances type-II collagen deposition over four weeks, while simultaneously reducing contraction. This combined effect suggests that Fasudil helps maintain defect fill while simultaneously promoting cartilage regeneration.
Clinical Relevance: Understanding the roles of the extracellular matrix organization and cell contractility in the early microfracture environment may provide insight into material-based or pharmacological treatments for improving the outcomes of microfracture.
Table of Contents
Table of Contents
1. Introduction
1.1 Motivation
1.2 Microfracture
1.3 Fibrinogen Overview
1.4 The Rho/ROCK Pathway, Fasudil, and TGF-β3
1.5 Objectives and Significance
2. Materials and Methods
Aim 1: Developing a Fibrin-based Microfracture Clot Model
2.1.1 Thrombin-Mediated Gel Mechanics
2.1.2 Thrombin-Mediated Gel Contraction
2.1.3 Microscale Fiber Formation
2.1.4 Thrombin-Dependent Gene Expression
Aim 2: Microscale Cell Behavior
2.2.1 Fibrin Densification
2.2.2 Cell Mechanics
2.2.3 Immunofluorescence
Aim 3: Macroscale Clot Behavior
2.3.1 Macroscale Clot Contraction
2.3.2 Histology
2.3.3 Gene Expression
2.3.4 Statistical Analysis
3. Results
Aim 1: Developing a Fibrin-based Microfracture Clot Model
3.1.1 Elastic Moduli of Fibrin Gels based on Thrombin Content
3.1.2 Thrombin-Dependent Gel Contraction
3.1.3 Microscale Fiber Formation
3.1.4 Thrombin-Dependent Gene Expression
Aim 2: Microscale Cell Behavior
3.2.1 Fibrin Densification
3.2.2 Cell Mechanics
3.2.3 Immunofluorescence
Aim 3: Macroscale Clot Behavior
3.3.1 Macroscale Clot Contraction
3.3.2 Histology Images and Interpretations
3.3.3 Gene Expression
4. Discussion
5. Conclusion
Supplementary Information
References
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File download under embargo until 24 May 2025 | 2024-04-09 11:21:28 -0400 | File download under embargo until 24 May 2025 |
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