Characterization of Cellular Response to a Novel Steady State Release of Hydrogen Peroxide at a Concentration Capable of Eradicating

One of the most prevalent forms of dental infections is periodontitis, which is characterized by accumulation of subgingival bacteria resulting in chronic inflammation. Mixed gram-positive and gram-negative microorganisms initiate the inflammatory response; however, matrix metalloproteinase (MMP) driven by the host response are the major drivers of tissue destruction in the periodontium. Pathogenic response to lipopolysaccharide (LPS) is known to recruit monocytes, macrophages and fibroblast. Consequently, this response triggers increased production of cytokines (IL-1, IL-6, IL-8, TNF-α). Current treatment of dental infection includes surgical reduction and systemic and local delivery of antibiotics. Chronic dental infections do not always respond to antibiotics and often cause side effects. Therefore, treatment of dental infections with low dose hydrogen peroxide (H2O 2) may offer a non-antibiotic alterative. Without effective sustained treatment aimed at eliminating the pathogens and mediators of the inflammatory response, ultimate destruction of periodontal structures can lead to tooth and/or dental implant loss. Therefore, the overall objective of this study was to determine if H2O2 delivered in a sustained delivery system of hydroxyapatite (HA) could prevent the formation of the biofilm and cellular host response without altering cell viability, function and morphology. The experimental design for RAW 264.7 cells entailed the assessment of functional capacity through the measurement of malondialdehyde (MDA), nitric oxide (NO), and glutathione (GSH). Additionally, structural evaluation was performed through morphological and proliferation methods. The functional activities of CRL-2014 cells were assessed by measuring MDA, GSH and protein and structural change. The glucose oxidase (GOX) catalase (CAT) system was used to provide 10 μM H2O2 and 20% oxygen in culture after reaching steady state concentration at 150 minutes. CRL-2014 cells + 1 x 105 S. aureus bacteria + 10 μM H2O2 were plated on Blood Agar Quad Plates and inspected and photographed for growth of bacterial colonies. H2O2 released from HA capsule in vitro was measured spectrophotometrically. Briefly, the results revealed that significant reductions in cell numbers and significant increase in NO and GSH suggest that RAW 264.7 cells are sensitive to low dose steady state concentration H2O2 (10 μM) and maintain capability to reduce oxidative stress damage to the cells via the redox system. CRL-2014 cell proliferation, morphology, intracellular and cell membrane integrity was not altered after treatment with H2O22 (10 μM). CRL-2014 cells treated with conditioned media from RAW 264.7 cells treated with 10 μM H2O2 did not result in alteration in cell proliferation, cell membrane integrity nor intracellular damage. Overall, the cells were able to maintain viable structural activities. Staphylococcus aureus was eradicated upon exposure to 10 μM H2O 2 after steady state concentration was reached at 480 minutes. Lastly, H2O2 was capable of releasing from a HA drug delivery system within 24 hours in vitro. Based on the overall findings of this study, we propose H2O2 may have potential as an antibiotic alternative in the treatment of early dental infections. Future studies are needed to determine alternative doses and chronic exposure to oxidative agents at low doses.