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Item Differentiation and Activity of Murine Derived Stromal Osteoblasts After Electromagnetic Wave Stimulation(2022) Wu, Jennifer L.; Spolnik, Kenneth; Bruzzaniti, Angela; Ehrlich, Ygal; Warner, NedIntroduction: Elimination of bacteria and active infection within an infected root canal system is one of the primary objectives of nonsurgical root canal treatment. One of the measures of successful root canal treatment is subsequent bone healing of periapical lesions caused by previous infection. A previous study by Yumoto et al. showed that electromagnetic wave stimulation can increase proliferation of osteoblastic cells with no cytotoxicity, and it can also up-regulate growth factors such as vascular endothelial growth factor and platelet-derived growth factor.18 They also showed increased proliferation of an immortalized osteoblastic MC3T3-E1 cell line 3 days following electromagnetic stimulation (EMS).18 Previously, Pauly et al. found increased alkaline phosphatase (ALP) activity with 10 mA EMS application to primary murine calvaria-derived osteoblastic cells with 5 pulses at 1 second per pulse, but no significant differences were found for MTS proliferation nor mineral deposition compared to a negative control group.82 Optimization of the different variables including post-treatment incubation time, current delivery, and number of pulses per treatment may be necessary to improve osteogenic activity. The use of mesenchymal stem cells from murine bone marrow may also offer a physiologically relevant model for osteoblastic regeneration of periapical lesions. Objectives: The goal of this study was to investigate and optimize the effects of electromagnetic wave stimulation (EMS) on murine bone marrow mesenchymal stem cells (MSCs) by evaluating the proliferation and differentiation of the cells after exposure to different EMS treatment regimens. Materials and Methods: 5 x104 stromal osteoblasts (SOBs) were cultured in 24-well plates in α-MEM containing 10% fetal bovine serum. Cells were then subjected to pulsed EMS treatments of 1 mA, 10 mA, and 50 mA. EMS was generated using an electromagnetic apical treatment (EMAT) device created by J. Morita MFG Corp. Proliferation was assessed via MTS assay 1 days after treatment. For osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media, and SOBs were cultured for 14 days. Afterwards, alkaline phosphatase (ALP) activity and Alizarin-red S mineral deposition were quantified as measures of osteoblast activity. Cells grown in osteogenic media without EMS treatment served as the negative control. Results: Although MSC proliferation was unaffected by different EMS treatment regimens, 50 mA EMS resulted in a decrease in ALP activity and mineral deposition by osteoblasts. Conclusions: Our findings suggest bone healing by EMS may involve a different cellular mechanism, that is not reproduced in vitro in our studies. Utilizing different amperage and EMS regimens may improve osteogenic differentiation.Item Use of electromagnetic stimulation on an Enterococcus faecalis biofilm in root canal treated teeth in vitro(2019) Kindler, Justin K.; Spolnik, Kenneth J.; Duarte, Simone; Gregory, Richard L.; Ehrlich, Ygal; Bringas, Josef S.Introduction: Nonsurgical root canal therapy procedures aim to reduce the total microbial load within an infected root canal system through chemomechanical debridement of the root canal system via instrumentation in conjunction with an antibacterial irrigating solution. The most commonly used irrigant is sodium hypochlorite, often at concentrations toxic to human cells. Electromagnetic wave irradiation is a novel method of disinfection that has been shown to be bactericidal against planktonic microorganisms in solution, but its efficacy against an established biofilm is unknown. Pilot studies have demonstrated a synergistic killing effect with sodium hypochlorite through a process termed electromagnetic stimulation (EMS). If confirmed, lower concentrations of the current gold standard of 6.0-percent sodium hypochlorite could be used to irrigate infected root canals during endodontic treatment, resulting in less toxicity to human cells. There are also regenerative implications as EMS could be used to disinfect the root canals of immature teeth using 1.5-percent sodium hypochlorite, as recommended by the American Association of Endodontists. Objectives: The purpose of this in-vitro study was to evaluate the anti-biofilm effect of EMS against an established biofilm of Enterococcus faecalis. Materials and Methods: Single rooted teeth were cut to a standardized length (12 mm) and instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with E. faecalis, which grew for two weeks to form an established biofilm. There were five treatment groups: 1) 6.0-percent sodium hypochlorite; 2) 1.5-percent sodium hypochlorite; 3) 1.5-percent sodium hypochlorite with EMS; 4) 0.9-percent saline with EMS and 5) 0.9-percent saline. Samples were collected, plated, and incubated for two days. The number of CFUs/mL was determined and converted to log10. The effect of treatment group on bacterial counts was made using Wilcoxon Rank Sums Test. One sample per group was scored and split for confocal imaging. Null Hypothesis: Teeth treated with EMS in combination with 1.5-percent sodium hypochlorite or 0.9-percent saline will not demonstrate a significant anti-biofilm effect in comparison to those treated with 6.0-percent sodium hypochlorite alone. Results: 0.9-percent saline and 0.9-percent saline with EMS were significantly higher than 6.0-percent NaOCl, 1.5-percent NaOCl, and 1.5-percent NaOCl with EMS. 0.9-percent saline was significantly higher than 0.9-percent saline with EMS. The three groups that included treatment with NaOCl were not significantly different from each other. Confocal imaging confirmed the CFU findings. Conclusion: Because there was no growth in any of the NaOCl groups, the null hypothesis cannot be rejected. However, there was an antibiofilm effect when comparing the two saline groups, demonstrating that EMS has an antibiofilm effect. Future studies should focus on determining what concentration of NaOCl is most effective in combination with EMS.