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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 4  |  Page : 277-282

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


1 Private Practice, Murcia, Spain
2 Department of Implant and Biomaterial Research, Fundación Corazón de Jesús, San Juan, Argentina
3 Private Practice, New Delhi, India
4 Department of Orthodontics, Faculty of Medicine, University of Oviedo, Asturias, Spain

Date of Submission20-Jun-2021
Date of Acceptance04-Jul-2021
Date of Web Publication08-Oct-2021

Correspondence Address:
Josť Luis Calvo-Guirado
Mozart street n°4, 1 Floor C-D Zip code 30002, Murcia
Spain
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijds.ijds_79_21

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  Abstract 


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.

Keywords: Autologous graft, bone grafts, dentin graft, dentin grinder disinfection, graft, ground teeth, human teeth, particulate dentin graft, tooth extraction


How to cite this article:
Calvo-Guirado JL, Garcés-Villalá MA, Mahesh L, De Carlos-Villafranca FA. Effectiveness of chemical disinfection in discarding pathogenic bacteria of human particulate tooth graft: An In vitro study. Indian J Dent Sci 2021;13:277-82

How to cite this URL:
Calvo-Guirado JL, Garcés-Villalá MA, Mahesh L, De Carlos-Villafranca FA. Effectiveness of chemical disinfection in discarding pathogenic bacteria of human particulate tooth graft: An In vitro study. Indian J Dent Sci [serial online] 2021 [cited 2021 Oct 19];13:277-82. Available from: http://www.ijds.in/text.asp?2021/13/4/277/327816




  Introduction Top


Utilizing extracted human teeth as an immediate source for autologous graft to preserve and augment the alveolar ridge is becoming a standard clinical procedure due to the availability of efficient chairside tooth processing technology.[1],[2],[3],[4],[5],[6] In principle, autologous dentin tissue has inherent qualities of autologous bone and presents a negligible risk of exposing patients to disease transfer or contaminants. The material has the potential of becoming the gold standard for socket preservation. However, some concern is evident regarding bacteria that already reside in the biofilm on the extracted tooth. It is still prudent to attempt to clean as much of the bacteria as possible by mechanical cleaning of the tooth following the extraction using a bur or an ultrasonic scaler.[7],[8] In addition to mechanical removal of the biofilm and contaminants, some authors proposed to further disinfect the dentin graft using different chemical agents that are employed in root canal disinfection therapies.[9],[10],[11],[12],[13] Kim et al. reported that root blocks that were disinfected with 1% of chlorhexidine for 10 min enabled their safe use in grafting bone defects, while others used ethanol.[14] Some authors used acid treatment for desmineralizing denting graft also used for bacteria reduction.[3],[4] Binderman et al. suggested to cleanse and disinfect the autologous dentin particulate before grafting by chemical sterilization using sodium hydroxide 0.5M plus 20% ethanol (Dentin Cleanser, KometaBio Inc., NJ, USA) for 5 min followed by two rinses of the particulate in PBS. This 8-min process is said to render the tooth particulate bacteria free.[6] Calvo-Guirado et al. followed Binderman technique in order to use those disinfected teeth as bone graft in postextraction sockets.[15],[16],[17]

The objective of this study was to determine the antibacterial activity of dental disinfectant agents during processing of extracted teeth for immediate grafting of the extraction sites. We shall determine the antibacterial activity of (a) Dentin Cleanser 0.5M NaOH and 20% ethanol, (b) 10% of ethylenediaminetetraacetic acid (EDTA), (c) 10% citric acid, and (d) phosphate-buffered saline (PBS) when applied to bacterially contaminated particulate dentin.


  Materials and Methods Top


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. The patient signed an informed consent form to donate his teeth for use in this in vitro study. The teeth were cleaned using straight fissure carbide burs with water irrigation, removing soft tissue, calculus debris, and biofilm and then they were dried with an air syringe. After being cleaned and dried, the teeth were decoronated using a carbide tungsten bur in order to separate the crown from their root [Figure 1]. Then, roots and crowns were pulverized separately using the Smart Dentin Grinder® device (KometaBio Inc., Cresskill, NJ, USA), sieved into specific size ranges, and deposited into four separate glass containers. In this study, we have used two tooth particle sizes of 400–600 and of 800–1200 microns. The separation of crown particulate from root particulate was done in order to examine the effect of disinfectants on dentin versus dentin and enamel mix.
Figure 1: Human roots (a) and crowns (b) inside the Smart Dentin Grinder chamber

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The root and crown particulate source materials were then divided into four different groups, and in addition, a sample of human bone marrow representing cellular soft tissue was added for comparison. The groups are:

  • Group A: 800–1200 μm enamel + dentin
  • Group B: 400–600 μm enamel + dentin
  • Group C: human marrow tissue
  • Group D: 800–1200 μm dentin
  • Group E: 400–600 μm dentin.


All the particulate dentin and dentin–enamel mix were then autoclave sterilized for 15 min at 121°C. A sample from each group was put into brain–heart infusion broth and incubated for 24 h at 37°C to confirm sterility. Each of the five groups, ground teeth (n = 4) and marrow (n = 1) were further divided into three subgroups [Figure 2]. At that time, all 15 samples were inoculated, each group with 10 ml of culture medium that included one of the three types of bacteria, Escherichia Coli, Porphyromonas gingivalis, and Enterococcus faecalis for 1 h at room temperature and then incubated for 24 h in 370° C [Figure 3]. The kits of bacteria were KWIK-STIK™ 2 Pack Catalog No. 0495P of E. coli derived from ATCC® 35218, KWIK-STIK™ 2 Pack Catalog No. 0912P of P. gingivalis derived from ATCC® 33277™*, and KWIK-STIK™ 2 Pack Catalog No. 0366P E. faecalis derived from ATCC® 29212™*. Each KWIK-STIK unit contains a lyophilized microorganism pellet, an ampoule of hydrating fluid, and an inoculating swab. By putting the fluid into lyophilized bacterial pellet, the specific bacterial strain is dissolved and ready for inoculation. After incubation, the infected particulate dentin, dentin–enamel mix, and marrow tissue were transferred into plates with nutrient agar, Columbia blood agar (VWR International Eurolab, SL, Llinars del Vallés, Barcelona, Spain), and Schaedler agar with sheep blood (Remel, Lenexa, KS, USA) + Hemin Vitamin K plates. Each of 15 agar plates was manually infected a second time with one of the three bacteria [Figure 3] and [Figure 4]. The inoculation of each of bacteria onto the agar plate was performed by using the inoculating swab. After inoculating, all 15 plates were incubated in an oven at 37°C under low pressure and oxygen flow, for 48 h for the E. coli and E. faecalis and 72 h for the P. gingivalis, respectively [Figure 4].
Figure 2: (a) 800–1200 um enamel particulate; (b) 400–600 um enamel particulate; (c) Marrow human bone pieces; (d) 800–1200 um dentin particulate; (e) 400–600 um dentin particulate

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Figure 3: Petri dishes with tooth particles (a); Teeth particles contaminated (b); Big tooth particles surrounded by bacteria (c)

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Figure 4: Dentin particles contaminated with Porphyromonas gingivalis, Escherichia coli, and Enterococcus faecalis

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The tip of the pipette was replaced after each disc impregnation. The following solutions were used for each of the discs [Figure 5]:
Figure 5: The following solutions were used for each of the discs: (a) KometaBio Dentin Cleanser – 0.5N Sodium hydroxide and 20% ethanol solution; (b) Ethylenediaminetetraacetic acid; (c) Phosphate-buffered saline; (d) Citric acid 10%

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After an incubation period, paper discs impregnated with each of the cleansing agents were placed on the infected particulate [Figure 6]. Each of the blank sterile paper discs was impregnated with one of the cleansing agents using a sterile pipette in the amount of 5 ul.
Figure 6: Three bacteria used in the study, (a and b) Germ 1 Escherichia coli; (c and d) Germ 2 Enterococcus faecalis; (e and f) Germ 3 Porphyromonas gingivalis

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The inhibition halo method was used to assess the sensitivity of the cleansing product, as shown in [Figure 6]. The measurement of inhibition halos is a technique used in antibiogram to determine the susceptibility of the bacterium against antimicrobial agents such as oral antiseptics.[18],[19]

The halo inhibition samples were measured after 24, 48, and 72 h of incubation (37°C), with the help of a millimeter gauge provided by the Laboratory of Bacteriology and Mycology of the Catholic University of Murcia. The samples were observed with the naked eye at 30 cm away from the eyes. To measure the inhibition halos, the Petri dishes were placed on a white grid paper background with a millimeter interval grid. The grid was then aligned with the center of each blank disc so that the diameter of each halo could be easily measured through the center of the disc. Measurement was taken from one side of the ingestion zone to the other side of it [Table 1]. The bigger the halo effect, the more sensitive the bacteria is to the cleansing product, or the more effective the disinfectant is [Figure 7]. We measured the bacterial halo inhibition diameters at 24, 48, and 72 h and correlated these measurements to bacterial sensitivity by following the Duraffourd scale. For the interpretation of qualitative results, the guidelines by Duraffourd 1983 were taken as a reference.[20] These are:
Figure 7: Method of measuring the size of the halos

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Table 1: Bacterial inhibition halos diameter (mean±standard deviation)

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  • Negative (−) halo less than or equal to 8 mm in diameter
  • Sensitive (sensitive = +) halo from 9 to 14 mm
  • Very sensitive (very sensitive = ++) halo from 15 to 19 mm
  • Highly sensitive or positive (H. S. = +++) if it was equal to or >20 mm.


Statistical analysis

Statistical analysis was performed using PASW Statistics v. 18.0.0 software (SPSS)(Armonk, New York 10504-1722 United States of América). 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 Top


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 [Table 1], while citric acid was not effective according to the Duraffourd[20] 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 [Table 2].
Table 2: Kolmogorov-Smirnov and Shapiro-Wilk tests

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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 [Table 3].
Table 3: Different cleansers for crushed teeth in order to disinfect the particles

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  Discussion Top


The present study reveals that the Dentin Cleanser that consists of sodium hydroxide and ethanol is most effective in eliminating the pathogenic bacteria of the biofilm of tooth surfaces. Sodium hydroxide solution is being used as a cleansing agent because of its defatting properties in many medical applications. It is also effective in degrading proteins and nucleic acids as well as inactivating most viruses, bacteria, yeasts, fungi, and endotoxins. It is, therefore, considered a potent disinfection agent, as documented in several studies.[21],[22],[23] Thalhimer and Palmer reported that sodium hydroxide showed marked germicidal activity even in a dilution of 1: 2500,[21] while Olitsky and Boëz reported that sodium hydroxide was efficient against viruses.[24] The second active ingredient in the Dentin Cleanser solution, ethanol, is known for its denaturation action of proteins, effectiveness against a wide spectrum of microbial species. It is safer than other disinfectants because ethanol easily evaporates, and the human body can also metabolize ethanol. Altogether, the Dentin Cleanser is both a strong cleansing and disinfecting agent being able to penetrate and discard the biomass in the dentin tubules, exposing the clean surface of mineralized dentin matrix.

On the other hand, EDTA is a chelating agent that has been shown to remove bacteria by binding Mg2+ and Ca2+ ions from the outer cell wall of bacteria and eliminate biofilm colonization and proliferation by reducing their adhesion to particulate surfaces.[25],[26] The antimicrobial effects of EDTA have been demonstrated for a range of clinical microorganisms that include Gram-negative and Gram-positive bacteria, yeasts, ameba, and fungi. Furthermore, EDTA as a cation chelator of calcium, magnesium, and zinc is inactivating metabolic and energy functions of bacteria, thus inhibiting their growth and function. Although these two agents are both effective in eliminating bacteria, as we show in this in vitro study, we believe that in the context of using these agents on dentin particulate, the two work differently. The Dentin Cleanser (sodium hydroxide plus ethanol) exposes the dentin to a high alkaline environment that breaks down proteins and removes nucleic acids as well as cleanses the biomass from dentin particulate surfaces. EDTA, on the other hand, as a chelating agent reduces the binding of bacteria to dentin surface and their growth. Moreover, EDTA is known to dissolve hydroxyapatite dentin mineral by chelating its calcium, thus exposing its organic collagenous matrix. In contrast, 10% of citric acid, that is able to dissolve the dentin mineral, was not effective in eliminating the pathogenic bacteria.


  Conclusions Top


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 the 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.

Ethical clearance

The Ethical Comitee 7212/2018. San Antonio Catholic University of Murcia.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Kim YK, Yun PY, Um IW, Lee HJ, Yi YJ, Bae JH, et al. Alveolar ridge preservation of an extraction socket using autogenous tooth bone graft material for implant site development: Prospective case series. J Adv Prosthodont 2014;6:521-7.  Back to cited text no. 1
    
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
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