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 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 activities under hard polydimethylsiloxane were significantly higher than those under soft and medium hard polydimethylsiloxane, and the activation of Wnt/β-catenin pathway was stronger than that under soft and medium hard polydimethylsiloxane. Immunofluorescence staining results showed that hard polydimethylsiloxane induced the shortening of primary cilia and the increase of YAP protein-positive cells. After interfering with YAP protein expression by siRNA, the inter-group differences in YAP, phosphorylated GSK-3β, β-catenin protein expression, and c-myc and CCND1 transcriptional activities disappeared, accompanied by the disappearance of primary cilia length differences. The results show that the hardness of the extracellular matrix directly regulates the proliferation activity of bone marrow stromal stem cells through the new mechanism of YAP protein/primary cilia.

Key words: polydimethylsiloxane, stiffness, bone marrow stromal stem cell, proliferation, YAP protein, primary cilium, β-catenin, Wnt pathway, extracellular matrix