Peptide-Enhanced Calcium Phosphates (CaPs) for the Management of Medication-related Osteonecrosis of the Jaw (MRONJ)

Abstract

Medication-related osteonecrosis of the jaw (MRONJ) is a serious condition associated with antiresorptive medications such as bisphosphonates (BPs). It involves exposed necrotic bone, often accompanied by microbial colonisation, which contributes to its pathogenesis. Calcium phosphate (CaP) materials can reduce local BP concentrations, while antimicrobial peptides (AMPs) are promising due to broad spectrum activity. This thesis aimed to develop a local therapeutic delivery system for MRONJ management by functionalising synthetic ReproBone (RB) calcium phosphate discs to induce antimicrobial activity through covalent immobilisation of AMPs. The antimicrobial, osteogenic, and biocompatibility properties of peptides KR-12, HHC-36, and KRSR, as well as peptide coated discs, were investigated. Swabs from MRONJ patients at Charles Clifford Dental Hospital (REC: 20/WS/0165) were analysed by 16S rRNA sequencing, revealing diverse mixed communities of microorganisms. Four species, Actinomyces oris, Prevotella buccae, Fusobacterium nucleatum, and Cutibacterium acnes, were chosen for antimicrobial testing as they are frequently isolated and well documented in MRONJ. KR-12 and HHC-36 displayed antimicrobial activity, while KRSR, without antimicrobial properties, acted as an osteogenic control. KR-12 also showed time dependent inhibition in mixed cultures. KR-12 and KRSR were biocompatible with MG-63 cells, whereas HHC-36 was cytotoxic at high concentrations. Osteogenic effects of KR-12 and KRSR, assessed by ALP activity, indicated enhanced osteogenic potential under osteogenic conditions. RB discs were functionalised via plasma polymerisation of acrylic acid (ppAAc) to introduce carboxylic groups, confirmed by toluidine blue O (TBO) assay, FTIR, andIV XPS. Peptides were covalently attached using EDC/NHS chemistry, verified by FTIR and XPS. All antimicrobial and biocompatibility experiments were performed in triplicate, while osteogenic assays with peptide treatments were conducted in duplicate. Where applicable, representative images and graphs are presented for each experiment, with similar results obtained in all repeats. KR-12 and KRSR coated RB discs were biocompatible with MG-63 cells. KR-12 coated discs exhibited antibacterial effects against MRONJ isolates through dose dependent inhibition. TEM imaging showed bacterial morphological disruption after exposure, supporting the potential of KR-12 peptide functionalised RB discs as future MRONJ management strategies.