Using an electrospinning technique, we constructed composite nanofibrous membranes containing a copolymer of L-lactide and glycolide (PLGA, ratio 85:15) and 33% wt.% of nanodiamond particles. The number of initially adhering human osteoblast-like MG 63 cells on day 1 after seeding, their spreading and subsequent growth were similar on both types of membranes. However, higher cell numbers on day 3 and 7 after seeding and a larger cell spreading area were found in the cells in the control polystyrene cell culture dishes. Nevertheless, the composite PLGA-ND membranes provided relatively good support for colonization with bone-derived cells; thus this material is promising for bone tissue engineering.
Production of inducible antimicrobial peptides offers a first and rapid defense response of epithelial cells against invading microbes. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide induced in various epithelia upon extracellular as well as intracellular bacterial challenge. Nucleotide-binding oligomerization domain protein 2 (NOD2/CARD15) is a cytosolic protein involved in intracellular recognition of microbes by sensing peptidoglycan fragments (e.g. muramyl dipeptide). We used luciferase as a reporter gene for a 2.3-kb hBD-2 promoter to test the hypothesis that NOD2 mediates the induction of hBD-2. Activation of NOD2 in NOD2-overexpressing human embryonic kidney 293 cells through its ligand muramyl dipeptide (MDP) induced hBD-2 expression. In contrast, overexpression of NOD2 containing the 3020insC frame-shift mutation, the most frequent NOD2 variant associated with Crohn disease, resulted in defective induction of hBD-2 through MDP. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for NOD2-mediated induction of hBD-2 through MDP. Moreover, the NF-kappaB inhibitor Helenalin as well as a super-repressor form of the NF-kappaB inhibitor IkappaB strongly inhibited NOD2-mediated hBD-2 promoter activation. Expression of NOD2 was detected in primary keratinocytes, and stimulation of these cells with MDP induced hBD-2 peptide release. In contrast, small interference RNA-mediated down-regulation of NOD2 expression in primary keratinocytes resulted in a defective induction of hBD-2 upon MDP treatment. Together, these data suggest that NOD2 serves as an intracellular pattern recognition receptor to enhance host defense by inducing the production of antimicrobial peptides such as hBD-2.
Carbon materials including carbon nanoparticles, such as nanographite, graphene and graphenic materials, and carbon nanotubes are known to be highly hydrophobic. Oxidation treatments are widely used as the best methods to improve their affinity in a liquid medium or a polymer matrix so that they can be dispersed, handled and processed. Here, we have applied eight different oxidation treatments in order to graft oxygen-containing functional groups at the surface of polyhedral graphitic particles synthesized by arc discharge from graphite, also called astralenes. The used functionalization approaches include both standard chemical attack by strong oxidants and radical functionalization of the sp2 network by direct C[double bond, length as m-dash]C bond opening. Commonly efficient functionalization methods were unsuccessful to functionalize astralenes while radicals generated from arylhydrazine could lead to functionalization of the outer surface of astralenes. The occurrence of functionalization could be shown by TGA coupled with MS and XPS. The reported method represents the first example of functionalization of astralenes. The efficiency of the applied functionalization methods is discussed considering the chemical reactivity of different carbon nanomaterials including graphene and carbon nanotubes.
Artificial materials such as dental implants are at risk of bacterial contamination in the oral cavity. Human beta defensins (HBDs), small cationic antimicrobial peptides that exert a broad-spectrum antibacterial function at epithelial surfaces and within some mesenchymal tissues, could probably help to reduce such contamination. HBDs also have protective immunomodulatory effects and have been reported to promote bone remodeling. The aim of this study, therefore, was to investigate the influence of recombinant HBD-2 on the proliferation and survival of cells in culture.Human mesenchymal stem cells (hMSCs), human osteoblasts, human keratinocytes (control), and the HeLa cancer cell line (control) were incubated with recombinant HBD-2 (1, 5, 10, or 20 μg/mL). Cell proliferation and cytotoxicity were evaluated via a water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase assays, respectively.HBD-2 was not toxic in any tested concentration to hMSCs, osteoblasts, keratinocytes, or HeLa cells. Furthermore, proliferation of hMSCs and osteoblasts increased after treatment with HBD-2 at all tested concentrations, and keratinocyte proliferation increased when treated at 20 μg/mL. In contrast, HeLa cancer cells were not affected by HBD-2 as tested.HBD-2 is not only biocompatible but also promotes proliferation of hMSCs, osteoblasts, and keratinocytes in culture. Further investigation of HBD-2 functional surface coating of artificial materials is recommended.
