Role of LL37-Au NPs in Antimicrobial Activity and Dental Application

The aim of this thesis is to study the strategy for revitalization of damaged teeth, by eliminating infections and regenerating the dentine-pulp complex instead of traditional root canal treatment, such as the obturation of the root canal with biologically inert materials and the use of sodium hypochlorite (NaOCl), as an endodontic irrigant and antimicrobial agent. NaOCl can induce potential complications ranging from permanent bleaching of clothes to serious soft tissue damage. The main problem for a dental pulp damaged is linked to bacterial contamination of the root canals. Therefore in this work, a multifunctional nanoformulation has been developed as potential antimicrobial and differentiation agent to differentiate dental stem cells to odontoblast-like cells.The Human Cathelicidin LL37 shows important antimicrobial and pro-regenerative properties. Peptide can be chemically immobilized on nanoparticles in order to increase its stability, decrease toxicity, improve antimicrobial properties. Gold nanoparticles (AuNPs) have good compatibility with human body, low toxicity, small size and stability and it is easy to modify their properties, immobilize high concentrations of peptide per surface area. In this project, LL37-conjugated AuNPs have been prepared by a one step procedure. In the first part of the work, the antimicrobial activity of the LL37-Au NPs has been evaluated against Gram-negative (E. coli and E. coli ML-35p) and Gram-positive (S. aureus, S. epidermidis, E. faecalis) bacteria using Broth Microdilution Antibacterial Assay and spread plate technique. In order to improve the antimicrobial activity of the LL37 peptide a green laser was used.The thermal energy generated by AuNPs after exposure of laser light helps to damage bacterial membranes and allows the internalization and bactericidal action of LL37-AuNPs.In the second part of the work, it has been shown the effect of LL37-AuNPs on stem cells from the apical papilla (SCAPs). The apical papilla tissue is found in the root of an immature permanent tooth.They have the capacity to differentiate into adipocytes and osteoblasts/odontoblasts in vitro condition. SCAPs as well as mesenchymal stem cells (MSCs) have the following characteristics: self-renewal, proliferation, migration, differentiation and immunosuppression, they can be used in immunotherapy and in regeneration of dental tissues, neural, bone and vascular tissues.To determine potential cytotoxic effects of the AuNPs and peptide LL37, the CellTiter-Glo Luminescent Cell Viability Assay was conducted. The differentiation of SCAPs was induced by odontogenic differentiation media, which contains dexamethasone and ꞵ-glycerophosphate. Alkaline phosphatase and Alizarin Red staining were used to study the odontogenic differentiation of SCAPs. To investigate the effects of LL37, AuNPs and LL37-AuNPs on the cellular proliferation of SCAPs, an In Cell Analyzer was used and the expression of epidermal growth factor (EGF) receptor on the surface of SCAPs was shown using flow cytometry. Moreover, LL37-AuNPs and Au-NPs were deposited on electrospun nanofibers of gelatin and antimicrobial properties have been tested. Further studies are necessary to understand how to optimize the parameters used in this work with the evidence that multifunctional nanoformulation could be further applied in clinical therapies.