Mechanical Trapping of the Cell Nucleus into Microgroove Concavity but not on Convexity Induces Cell Tissue Growth and Vascular Smooth Muscle Differentiation

Cardiovascular System Medicine Life Sciences Bioengineering Cell Biology
cell biomechanics mechanobiology vascular smooth muscle cell differentiation microfabrication

Academic Background

Vascular Smooth Muscle Cells (VSMCs) in the normal aortic wall regulate vascular contraction and dilation. However, under pathological conditions, VSMCs transition from a contractile phenotype to a synthetic phenotype and actively participate in the remodeling of the aortic wall. Although many in vitro studies have reported the mechanisms of VSMC differentiation, the mechanical environment of in vitro culture conditions significantly differs from that of the in vivo aortic wall. In vivo, VSMCs exhibit an elongated morphology and align circumferentially along the vessel wall, whereas in vitro, VSMCs spread randomly and form irregular shapes, often leading to dedifferentiation. Therefore, developing a cell culture model that mimics the mechanical environment of the in vivo aortic wall is crucial for elucidating the mechanisms of VSMC differentiation.

Source of the Paper

This paper was co-authored by Kazuaki Nagayama and Naoki Wataya from the Micro-Nano Biomechanics Laboratory, Department of Mechanical Systems Engineering, Ibaraki University, Japan. The paper was published online on October 22, 2024, in the journal Cellular and Molecular Bioengineering.

Research Process and Results

1. Fabrication of Microgrooved Substrates and Establishment of Cell Culture Model

The research team first fabricated polydimethylsiloxane (PDMS)-based microgrooved substrates with groove widths of 5, 10, and 20 micrometers and a uniform depth of 5 micrometers. To mimic the elastic lamellar structure of the in vivo aortic wall, the researchers designed these microgrooved substrates and developed a method to coat cell adhesion proteins exclusively on the concave surfaces of the grooves. This method allowed the researchers to control the adhesion of VSMCs to the concave surfaces, thereby inducing cell elongation and alignment.

2. Morphological Analysis of Cell Nuclei

The researchers used confocal fluorescence microscopy to perform a detailed analysis of the morphology of VSMC nuclei. The results showed that VSMCs cultured on the 5-micrometer-wide microgrooved substrates exhibited significant nuclear elongation and volume reduction. The aspect ratio (length-to-width ratio) of the nuclei was highest in the 5-micrometer groove group, indicating that the nuclei were mechanically compressed by the sidewalls of the groove concavities, leading to significant morphological changes. Additionally, the density of intranuclear DNA increased significantly, suggesting that the DNA was condensed under mechanical compression.

3. Analysis of Cell Migration and Proliferation

To evaluate the effects of the microgroove concavities on VSMC migration and proliferation, the researchers conducted cell migration and proliferation assays. The results showed that VSMCs cultured on the 5-micrometer groove substrates exhibited significantly reduced migration speed and proliferation rates. In contrast, VSMCs on the 20-micrometer groove substrates showed higher migration speeds and proliferation rates. This suggests that the mechanical restriction of the nuclei in the 5-micrometer groove concavities inhibited cell migration and proliferation.

4. Analysis of VSMC Differentiation

The researchers further assessed VSMC differentiation by measuring the expression levels of smooth muscle α-actin (α-SMA) and calponin as differentiation markers. The results showed that VSMCs cultured on the 5-micrometer groove substrates exhibited significantly higher expression levels of α-SMA and calponin compared to other groups. This indicates that the mechanical restriction of the nuclei in the 5-micrometer groove concavities promoted VSMC differentiation.

Conclusion and Significance

The results demonstrate that mechanically trapping the cell nucleus in the microgroove concavities significantly inhibits VSMC migration and proliferation while promoting differentiation. This finding provides new insights into the mechanisms of VSMC differentiation and offers an important reference for developing cell culture models that mimic the mechanical environment of the in vivo vascular system. The study not only holds significant scientific value but also offers potential applications in the treatment of cardiovascular diseases and vascular tissue engineering.

Research Highlights

  1. Innovative Design of Microgrooved Substrates: The research team developed a method to coat cell adhesion proteins exclusively on the concave surfaces of the grooves, successfully mimicking the elastic lamellar structure of the in vivo aortic wall.
  2. Mechanical Regulation of Nuclear Morphology: The study revealed for the first time that mechanical compression of the nuclei in the microgroove concavities leads to significant changes in nuclear morphology and function, influencing cell migration, proliferation, and differentiation.
  3. Mechanism of Differentiation Promotion: The research found that mechanical restriction of the nuclei promotes VSMC differentiation by stabilizing intracellular polarity and cell-cell connections.

Other Valuable Information

The research team also suggested that future studies could combine mechanical stretch stimulation to further explore the detailed mechanisms of VSMC differentiation and dedifferentiation. Additionally, combining serum starvation culture with microgrooved substrates may have significant implications for vascular tissue regeneration.

This paper, through innovative experimental design and detailed data analysis, reveals the regulatory mechanisms of VSMC behavior under mechanical restriction of the nuclei in microgroove concavities, providing new insights and tools for cardiovascular research and tissue engineering.