Associate Professor of Chemistry Carnegie Mellon University, United States
Introduction: Bone fractures are the most frequent large organ, traumatic injury in humans, and some known as critical size bone defects are too large to heal on their own, requiring intervention. Current research in the treatment of critical size bone defects favors the development of biogradable, bioactive biomaterials that support and enhance regeneration of native bone over traditional metal implants and natural bone grafts. Graphene oxide (GO) makes a great candidate for this application because it is biocompatible, osteoinductive, strong, is safely and slowly resorbed by the body, and has a cheap, facile, and scalable synthesis. Its bioactivity can be further enhanced via functionalization with biomolecules such as peptides. Bone regeneration scaffolds frequently face challenges in retaining implanted and recruited cells, and promoting their survival, proliferation, and differentiation. Here, several short peptides RGD, DGEA, and KKGHK are bound to graphene oxide to improve cell adhesion, regeneration of bone tissue, and repair of the vascular network, respectively. Repairing the vascular network is crucial in ensuring tissue oxygen supply and therefore survival. This is accomplished through covalent chemical linkages as opposed to noncovalent association to allow for sustained retention of the peptides and therefore sustained bioactivity of the material. In vitro assays demonstrate the desired biological effects of these conjugate materials.
Materials and
Methods: Graphene oxide (GO) was synthesized from graphite via the Hummer’s method, and side chain-protected RGD, DGEA, and KKGHK peptides were synthesized via solid phase peptide synthesis. Basal plane alcohols on GO were converted to carboxylic acids via a Johnson-Claisen rearrangement, and these were further converted to acyl chlorides using a strong chlorinating agent. The resulting acyl chloride graphene reacted with each peptide via nucleophilic attack by the N-terminus at the acyl chloride to form a covalent bond between the materials, and deprotection removed the amino acid side chain protecting groups. Analytical techniques including Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were used to confirm successful synthesis and characterize the peptide graphene materials. Cytocompatibility was assessed by exposing two murine cell lines to dispersions of the peptide graphene’s. The specific bioactivities of the peptide graphene’s were assessed by different in vitro assays based on their peptide component: fibroblast spreading for RGD, mesenchymal stem cell osteogenesis for DGEA, and endothelial cell angiogenesis for KKGHK.
Results, Conclusions, and Discussions: Successful synthesis of covalently bound peptide graphene’s is evidenced by new unique peaks in the FTIR spectra compared to reactants and nitrogen content in the elemental analysis from XPS. Thorough washing is employed after synthesis, and so no unbound peptide should remain in the final material. There is no nitrogen present in the graphenic materials prior to peptide conjugation, so any nitrogen must result from covalently conjugated peptide. In a two-day cytocompatibility test, the peptide graphene’s were found to have no significant detrimental impact on cell survival and proliferation, especially compared to unconjugated graphenic materials. The RGD peptide is derived from fibronectin and found in many other proteins, and is known to be a point of cell attachment. Covalently binding the RGD peptide to GO should improve cell adhesion and spreading on the surface of the material by allowing for the formation of more focal adhesions. Fibroblasts cultured on RGD-GO pellets were found to have a higher cytoplasm area and lower cytoplasm roundness compared to fibroblasts cultured on reduced GO pellets. The DGEA peptide is derived from collagen type I and has been found to promote osteogenesis in stem cells. Covalently binding DGEA to GO should enhance its inherent osteogenic property. Mesenchymal stem cells cultured on DGEA-GO were found to have higher alkaline phosphatase levels and increased mineralization compared those cultured on reduced graphene oxide, as determined by alkaline phosphatase and alizarin red assays. These markers indicate differentiation into osteoblasts. Additionally, viewing the cells on these surfaces via fluorescence microscopy and SEM showed better survival and spreading on DGEA-GO compared to reduced GO. The KKGHK peptide is derived from osteonectin and has been shown to promote new blood vessel formation and blood vessel sprouting (angiogenesis) in endothelial cells. KKGHK-GO should support angiogenesis compared to reduced GO. This was indeed found to be the case in proliferation, migration, and tube formation assays with human umbilical cord endothelial cells. In conclusion, covalently incorporating these peptides with GO improves its in vitro bioactivity profile in the content of regenerating tissue following a severe bone injury.
