Effectiveness of chemical disinfection in discarding pathogenic bacteria of human particulate tooth graft: An In vitro study

Introduction: Extracted teeth are utilized in dentistry as particulate autologous dentin for immediate grafting of the extraction site after mechanical cleaning and chemical disinfection. The objective of this study was to determine the effectiveness of 0.5M sodium hydroxide in 20% ethanol (Dentin Cleanser™) in eliminating three different types of pathogenic bacteria in comparison to ethylenediaminetetraacetic acid (EDTA) or citric acid. Seven naive extracted teeth were mechanically cleaned, dried, and sectioned to separate the crown from the roots. Each tooth was separately crushed using the Smart Dentin Grinder® device. The sterile particles of crown or root were subdivided into three equal-size groups where each was then contaminated and incubated in an oven at 37°C under low pressure and oxygen flow over 48 h for Escherichia coli (Group A) and Enterococcus faecalis (Group B) and over 72 h for Porphyromonas gingivalis (Group C), respectively. On each agar Petri dish, four paper discs, each loaded with one of the following solutions: Dentin Cleanser (sodium hydroxide plus ethanol), 10% EDTA, phosphate-buffered saline (PBS), or 10% citric acid, were placed in the safe distance for not interfering with disinfectant agent activity. All pathogenic bacteria were highly sensitive to Dentin Cleanser and EDTA disinfectant activity while citric acid or PBS exhibited low or no sensitivity. No difference in sensitivity was found between crown and root particulate or particle size. Our findings show that Dentin Cleanser is most effective in eliminating those pathogenic bacteria without demineralizing the particulate. Context: The experiment was done in the University Laboratory. Aims: The objective of this study was to determine the effectiveness of 0.5M sodium hydroxide in 20% ethanol (Dentin Cleanser™) in eliminating three different types of pathogenic bacteria in comparison to EDTA or citric acid, before tooth graft will be used as a biomaterial. Settings and Design: The study protocol was approved by the Catholic University of Murcia Ethics Committee (UCAM; registration number 6781; July 21, 2017). Seven human teeth were extracted from a 60-year-old patient due to advanced periodontal disease (two central upper incisors, one upper canine, one upper premolar, two lower molars, and one lower canine). The patient received no financial compensation for participating in this study. Materials and Methods: Seven naïve extracted teeth were mechanically cleaned, dried, and sectioned to separate the crown from the roots. Each tooth was separately crushed using the Smart Dentin Grinder® device (KometaBio Inc., Cresskill, NJ, USA). The particles were sieved to obtain particles ranging from 400 to 600 um and 800–1200 um in size, all sterilized using an autoclave. The sterile particles of crown or root were subdivided into three equal-size groups where each was then contaminated and incubated in an oven at 37°C under low pressure and oxygen flow over 48 h for E. coli (Group A) and E. faecalis (Group B) and over 72 h for P. gingivalis (Group C), respectively. Then, each subgroup was immersed in 15 agar Petri dishes and again each was inoculated with the same bacteria allowing full growth of bacteria. On each agar Petri dish, four paper discs, each loaded with one of the following solutions: Dentin Cleanser (sodium hydroxide plus ethanol), 10% EDTA, PBS, or 10% citric acid, were placed in the safe distance for not interfering with disinfectant agent activity. Statistical Analysis Used: Statistical analysis was performed using PASW Statistics v. 18.0.0 software (SPSS). One-way analysis of variance was applied for the comparison of the means for halos, assuming a level of significance of 95% (P < 0.05). Kolmogorov–Smirnov and Shapiro–Wilk tests were applied for normality. Results: The results of the present study show that the 0.5M sodium hydroxide with 20% ethanol also termed as Dentin Cleanser presented the widest diameter halo of free bacterial growth, most effective against P. gingivalis (15.76 ± 0.28), less effective against E. faecalis (13.21 ± 0.72) and E. coli (12.14 ± 0.12). Similarly, EDTA was effective in eliminating the same strains of bacteria, while citric acid was not effective according to the Duraffourd halo inhibition scale. PBS had no effect in inhibiting bacterial growth. Both the Dentin Cleanser and EDTA were significantly effective in inhibiting bacterial growth in comparison to PBS and citric acid. No significant differences were observed between the dentin particulate and the dentin–enamel mix particulate or human bone marrow related to the level of sensitivity measured for each of the disinfectants tested. Conclusions: The use of the Dentin Cleanser (sodium hydroxide plus ethanol) or EDTA 10% appears to be an effective disinfectant method of tooth particulate prior to use of such particulate as autologous grafting material. Although E. coli, P. gingivalis, and E. faecalis were found to be sensitive to the presence of these disinfectants, additional bacterial types should be further investigated.