Functionalized Graphene Nanoparticles Induce Human Mesenchymal Stem Cells to Express Distinct Extracellular Matrix Proteins Mediating Osteogenesis

Abstract

Steven D Newby,1,2 Tom Masi,3 Christopher D Griffin,4 William J King,4 Anna Chipman,1 Stacy Stephenson,3 David E Anderson,1 Alexandru S Biris,4 Shawn E Bourdo,4 Madhu Dhar1 1College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA; 2Comparative and Experimental Medicine, University of Tennessee, Knoxville, TN 37996, USA; 3University of Tennessee Graduate School of Medicine, Knoxville, TN 37996, USA; 4Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little Rock, AR 72204, USACorrespondence: Madhu DharCollege of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USATel [phone]Fax + 1865-974-5773Email [email protected] E BourdoCenter for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little Rock, AR 72204, USATel [phone]Fax [phone]Email [email protected]: The extracellular matrix (ECM) labyrinthine network secreted by mesenchymal stem cells (MSCs) provides a microenvironment that enhances cell adherence, proliferation, viability, and differentiation. The potential of graphene-based nanomaterials to mimic a tissue-specific ECM has been recognized in designing bone tissue engineering scaffolds. In this study, we investigated the expression of specific ECM proteins when human fat-derived adult MSCs adhered and underwent osteogenic differentiation in the presence of functionalized graphene nanoparticles.Methods: Graphene nanoparticles with 6– 10% oxygen content were prepared and characterized by XPS, FTIR, AFM and Raman spectroscopy. Calcein-am and crystal violet staining were performed to evaluate viability and proliferation of human fat-derived MSCs on graphene nanoparticles. Alizarin red staining and quantitation were used to determine the effect of graphene nanoparticles on osteogenic differentiation. Finally, immunofluorescence assays were used to investigate the expression of ECM proteins during cell adhesion and osteogenic differentiation.Results: Our data show that in the presence of graphene, MSCs express specific integrin heterodimers and exhibit a distinct pattern of the corresponding bone-specific ECM proteins, primarily fibronectin, collagen I and vitronectin. Furthermore, MSCs undergo osteogenic differentiation spontaneously without any chemical induction, suggesting that the physicochemical properties of graphene nanoparticles might trigger the expression of bone-specific ECM.Conclusion: Understanding the cell–graphene interactions resulting in an osteogenic niche for MSCs will significantly improve the application of graphene nanoparticles in bone repair and regeneration.Keywords: graphene nanoparticles, functionalized graphene, human mesenchymal stem cells, extracellular matrix, fibronectin, collagen I, osteogenic niche  

Keywords

• graphene nanoparticles
• functionalized graphene
• human mesenchymal stem cells
• extracellular matrix
• fibronectin
• collagen i
• osteogenic niche