Functionalized Graphene Microsheets for Skeletal Tissue Engineering

Incorporation of carbon nanostructures into biodegradable polymer scaffolding for skeletal tissue engineering has been shown to increase the polymer's mechanical strength and to support cell growth; however, carbon nanotubes (CNTs) and fullerenes are expensive as starting materials. Graphite, or individualized sheets called graphene, provides similar scale, properties, and reaction pathways as fullerenes and CNTs, but at a fraction of the cost. Objective: Functionalize the surface of graphene micro-sheets with a high-density coverage of biomolecules. Methods: Azido-phenylalanine (Phe-N3) was synthesized and used as our nitrene source. Graphene sheets were pre-prepared by a solvent assisted exfoliation method to create individualized sheets prior to the reaction. Thermal decomposition of the azide to the nitrene results in a [2+1] nitrene addition to the C-C double bonds. Results: By thermogravimetric analysis (TGA), the product was determined to be 69% phenylalanine (wt%) correlating to a 10 to 1 carbon to Phe ratio (C:Phe). X-ray photoelectron spectroscopy (XPS) was used to investigate the chemical composition of the product (70.1% C, 16.1 % O, and 13.3 % N) and to confirm the extent of functionalization (C:Phe 13:1). Transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirmed that this synthesis method resulted in few-layer (n < 5) graphene sheets. Conclusions: Graphene/graphite has been shown to be preferentially functionalized on its edges; however, our method resulted in very high density surface coverage of the basal plane with Phe. The azido group has been used as a protecting group for organic synthesis and will tolerate a wide variety of other functionalities. Therefore, almost any primary amine containing biomolecule or polymer can be easily converted to the corresponding azide and conjugated to graphitic surfaces to create diverse scaffolding materials. In addition to the covalent linkage, surface functionalization could improve non-covalent interactions with the polymer matrix and cells. Funded by Welch Foundation.