title: Substrate Coupling Strength of Integrin-Binding Ligands Modulates Adhesion, Spreading, and Differentiation of Human Mesenchymal Stem Cells.
abstract: Substrate stiffness has been shown to regulate the differentiation fate of human mesenchymal stem cells (hMSCs). hMSCs sense and respond to substrate rigidity by exerting traction forces upon the binding between integrins and integrin-specific ligands present on the substrate surface. However, in previous studies, integrin-specific ligands such as Arg-Gly-Asp (RGD) peptides are always grafted to the substrate by a permanent covalent bond. Whether the coupling strength of integrin-specific ligands on substrate will influence cell behaviors has not been explored. In this work, we have developed a facile platform to investigate the effects of varied coupling strength between the RGD peptide and the glass substrate on stem cell behaviors. Glass coverslips are decorated with positive charges by silanization using (3-aminopropyl) triethoxysilane (APTES) to immobilize negatively charged citrate-capped gold nanoparticles (cit-AuNPs) solely via electrostatic interactions. The monolayer of electrostatically immobilized cit-AuNPs is further conjugated with the thiolated RGD peptides through the sulfur-gold bond. The substrate coupling strength of the RGD peptides, which is dependent on the electrostatic interactions between the APTES-treated glass substrate and the cit-AuNPs, is simply tuned by changing the APTES dosage and, hence, the resultant positive charge density on the surface. A total of 0.5% and 12.5% of APTES are used to fabricate low-coupling-strength surfaces (namely, LCS0.5 and LCS12.5), whereas 25% and 50% of APTES are used to fabricate high-coupling-strength surfaces (namely, HCS25 and HCS50). Fluorescence microscopy shows that hMSCs spread well and form stable actin filamentous structure on HCS surfaces but not on LCS surfaces. Remarkably, hMSCs exhibit enhanced osteogenesis on HCS surfaces as revealed by the immunostaining results of multiple early osteogenic markers. These differential behaviors may be governed by Yes-associated protein (YAP), a mechanosensitive transcriptional regulator of stem cells. Our findings highlight the importance of the substrate coupling strength of integrin-binding ligands on regulating adhesion, spreading, and differentiation of hMSCs.
authors.affiliation: Department of Mechanical and Automation Engineering, ‡Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong , Shatin, New Territories, Hong Kong, China.
authors.forename: Chun Kit K
authors.initials: CK
authors.lastname: Choi
authors.affiliation: Department of Mechanical and Automation Engineering, ‡Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong , Shatin, New Territories, Hong Kong, China.
authors.forename: Yang J
authors.initials: YJ
authors.lastname: Xu
authors.affiliation: Department of Mechanical and Automation Engineering, ‡Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong , Shatin, New Territories, Hong Kong, China.
authors.forename: Ben
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Mechanical and Automation Engineering, ‡Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong , Shatin, New Territories, Hong Kong, China.
authors.forename: Meiling
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Department of Mechanical and Automation Engineering, ‡Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong , Shatin, New Territories, Hong Kong, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Mechanical and Automation Engineering, ‡Shun Hing Institute of Advanced Engineering, The Chinese University of Hong Kong , Shatin, New Territories, Hong Kong, China.
authors.forename: Liming
authors.initials: L
authors.lastname: Bian
mesh_terms: D002448:Cell Adhesion; D002454:Cell Differentiation; D006046:Gold / Q000737:chemistry; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008024:Ligands; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D053768:Metal Nanoparticles; D009842:Oligopeptides / Q000737:chemistry; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
chemical_list: D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; D006046:Gold; C047981:arginyl-glycyl-aspartic acid
keywords: RGD peptide coupling strength; cell adhesion; cell spreading; cell−substratum interactions; stem cell differentiation