Extracellular matrix stiffness affects the proliferation activity of bone marrow stromal stem cells

Abstract

BACKGROUND: In tissue engineering bone construction, the physical properties of the scaffold can directly affect the activity and repair effect of seed cells, among which extracellular matrix hardness is a key factor affecting seed cell proliferation activity. Primary cilia and YAP proteins have been shown to be classical mechanoreceptors and downstream transduction factors, which may directly mediate this mechanism. OBJECTIVE: To investigate the regulatory effect of extracellular matrix hardness on the proliferation activity of bone marrow stromal stem cells and the related mechanisms. METHODS: Bone marrow stromal stem cells were passaged and seeded under different hardness of polydimethylsiloxane extracellular matrix conditions (soft, median, and rigid) for culture. Cell proliferation activity was detected using CCK-8 assay. Transcriptional activity of proliferation genes c-myc and CCND1 was measured using qRT-PCR. Activation of Wnt/β-catenin pathway was evaluated using western blot assay. Primary cilia and YAP protein expression levels were evaluated by acetylated α-tubulin and YAP immunofluorescence staining. After passage, bone marrow stromal stem cells were inoculated on polydimethylsiloxane-based membranes of different hardness (soft and hard) for culture. Then siRNA was used to interfere with YAP protein expression. Western blot assay was used to detect YAP, phosphorylated GSK-3β, and β-catenin protein expression. qRT-PCR was used to detect the transcriptional activity of c-myc and CCND1. The length of primary cilia was analyzed after immunofluorescence staining of acetylated α-tubulin. RESULTS AND CONCLUSION: The cell proliferation activity, c-myc and CCND1 transcriptional activity under rigid polydimethylsiloxane conditions were significantly higher than those under soft and median hardness, and the activation of Wnt/β-catenin pathway was stronger. Immunofluorescence staining showed that rigid polydimethylsiloxane induced shortening of primary cilia and increased YAP-positive cells. After siRNA interference of YAP expression, the differences in YAP, phosphorylated GSK-3β, β-catenin protein expression, and c-myc and CCND1 transcriptional activity between groups disappeared, accompanied by the disappearance of primary cilia length differences. The results indicate that extracellular matrix stiffness regulates the proliferation activity of bone marrow stromal stem cells through a novel YAP protein/primary cilia mechanism.

1. Introduction

In tissue engineering bone construction, scaffolds loaded with cells serve as extracellular matrix components, influencing seed cell activity and repair outcomes. Recently, various novel scaffolds have been widely applied in bone tissue engineering repair [1-2], showing high potential value. Among these, scaffold stiffness can directly affect multiple activities of seed cells, including proliferation, migration, and differentiation [3-4], thereby precisely controlling cell behavior, representing an important research direction for enhancing bone repair capacity. Although numerous studies have addressed the regulation of cell activity by physical properties of the extracellular matrix, research on how matrix stiffness affects proliferation and differentiation of bone marrow stromal stem cells remains insufficient [5]. This study aims to reveal the underlying molecular mechanisms in this process.

Recent literature has confirmed that primary cilia and YAP protein/Hippo pathway can transduce mechanical signals/extracellular matrix stiffness signals, activating downstream pathways and affecting cell activity [6-7]. These two have interactive mechanisms: primary cilia can mediate YAP activity to influence inflammatory responses [8-9], and extracellular matrix stiffness can directly affect Wnt1 and β-catenin protein expression, regulating cell proliferation [3]. This study proposes that scaffold/extracellular matrix stiffness may regulate seed cell proliferation through a primary cilia/YAP signaling mechanism, activating the downstream Wnt/β-catenin pathway. These results will help elucidate the 'scaffold stiffness-cell activity' regulatory mechanism and provide new insights for tissue engineering bone construction and mechanical microenvironment design.