Microenvironmental Stiffness Regulates Dental Papilla Cell Differentiation: Implications for the Importance of Fibronectin–Paxillin−β-Catenin Axis

Abstract

The mechanical stiffness of substrates is recognized to be an important physical cue in the microenvironment of local cellular residents in mammalian species due to their great capacity in regulating cell behavior. Dental papilla cells (DPCs) play an important role in the field of dental tissue engineering for their stem cell-like properties. Therefore, it is essential to provide the suitable microenvironment by combining with the physical cues of biomaterials for DPCs to carry out the function of effective tissue regeneration. However, how the substrate stiffness influences the odontogenic differentiation of DPCs is still unclear. Thus, we fabricated poly(dimethylsiloxane) substrates with varied stiffness for cell behavior. Both cell morphology and focal adhesion were shown to have significant changes in response to varied stiffness. Paxillin, an important protein adapter of focal adhesion kinase protein, was shown to interact with both ectoplasmic fibronectin and cytoplasmic β-catenin by coimmunoprecipitation. The resultant changes of β-catenin by varied stiffness were confirmed by immunofluorescent stain and western blotting. Further, the higher quantity nuclear translocation of β-catenin and the less phospho-β-catenin on the stiff substrate were detected. This nuclear translocation in the stiff substrate finally led to an increased mineralization of DPCs relative to the soft substrate detected by Von Kossa and Alizarin Red stain. Taken together, this work not only points out that the substrate stiffness can regulate the odontogenic differentiation potential of DPCs via fibronectin/paxillin/β-catenin pathway but also provides significant consequence for biomechanical control of cell behavior in cell-based tooth tissue regeneration.

Compliant substratum modulates vinculin expression in focal adhesion plaques in skeletal cells

“…The role of FAK and paxillin in green has been elucidated in our previous data. 19,34 The grey parts are involved in the vinculin-cytoskeleton rearrangements but not in our study…”

“…33 Surface roughness could also influence cell functions, such as cell migration, cell proliferation and differentiation. 34 For hydrophilicity, Sun et al 35 systematically summarized and illustrated the biological processes influenced by the hydrophilicity from structural biointerface materials and smart biointerface materials. Menon et al 36 found that the migration of human bone marrow-derived mesenchymal stem cells (hBMSCs) was greatly enhanced when cells were seeded on the PDMS substrate with intermediate levels (1:20) and that the proliferation was increased when the cells were seeded on the PDMS substrate with a higher stiffness.…”

“…19 In dental papilla cells (DPCs), we found that paxillin interacts with both ectoplasmic fibronectin and cytoplasmic β-catenin signalling by direct binding and plays a vital role in DPC mineralization. 34 In chondrocytes, we have detected the importance of the RhoA/ROCK pathway in the maintenance of the chondrocyte phenotype, 43 but the specific biomechanical control mechanism is deficient. In addition, further elucidation of the modulation process of mechanosensing and mechanotransduction in other cells, such as osteoblasts and osteoclasts, in the skeletal system is needed.

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Effective Delivery of the CRISPR/Cas9 System Enabled by Functionalized Mesoporous Silica Nanoparticles for GFP‐Tagged Paxillin Knock‐In

“…Fibronectin, as the ligand for a dozen members of the integrin receptor family, could bind paxillin to achieve the transduction of environmental signals to the cell interior. [31] After plated on fibronectin, U2OS cells-localized focal adhesion sites displayed a punctate distribution with bright green fluorescence (Figure 7c). When staining the edited cells with monoclonal paxillin antibody and Alexa Fluor@647-conjugated goat anti-rabbit lgG (which was predicted to label both the GFP-tagged and untagged paxillin protein factions within the same cells), obvious colocalization of different fluorescence channels (paxillin, red and GFP, green) was observed in Figure 7c.…”

“…[29] Therefore, mutation in the PXN gene, as well as abnormal expression of paxillin protein, has been implicated in the progression of various cancers. [30,31] For this purpose, nuclear localization sequence (NLS)-conjugated MSNs were prepared, which could further immobilize the plasmids with high loading efficiency (50%). After pH-responsive polymer (poly(dimethyldiallylammonium chloride), PDDA) coating in water assisted by microfluidic technology, the positively charged nanocarrier can efficiently protect loaded plasmids, and subsequently it can enhance the cellular internalization and endosomal escape.…”