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Year : 2018  |  Volume : 21  |  Issue : 4  |  Page : 388-393
Comparative evaluation of coronal discoloration induced by two triple antibiotic revascularization protocols when used at varying depths of temporary sealing material at the end of varying time periods

1 Dr. D. Y. Patil Dental College & Hospital, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharastra, India
2 University of Michigan, Detroit, USA

Click here for correspondence address and email

Date of Submission23-Mar-2017
Date of Decision07-Mar-2018
Date of Acceptance19-Mar-2018
Date of Web Publication27-Jul-2018


Aim: The aim of this study is to evaluate and compare the coronal discoloration induced by two Triple antibiotic paste (TAP 1 and 2), when used with varying depths of 2 and 4 mm of temporary sealing materials at the end of 0, 3, and 6 weeks.
Objectives: (1) To evaluate coronal discoloration induced by TAP-1 when the depth of the temporary restorative material is 2 mm below cementoenamel junction at the end of 0, 3, and 6 weeks.
Materials and Methods: Forty extracted permanent maxillary central and lateral incisors or single-rooted tooth were collected and chosen for the study. They were then divided into two groups and each group was then subdivided into two subgroups.
Results: Both the triple antibiotic paste showed discoloration but it reduced with increase in thickness of temporary cement.
Conclusion: TAP 1 and TAP 2 both showed an increase in discoloration, greater discoloration was seen with TAP, containing minocycline. There was a steady change in the discoloration from 0 to 6th week. In both the groups, TAP 1 and TAP 2, there was a reduction in discoloration with increase thickness of the temporary restorative material.

Keywords: Discoloration; disinfection; revascularization; triple antibiotic paste

How to cite this article:
Jagdale S, Bhargava K, Bhosale S, Kumar T, Chawla M, Jagtap P. Comparative evaluation of coronal discoloration induced by two triple antibiotic revascularization protocols when used at varying depths of temporary sealing material at the end of varying time periods. J Conserv Dent 2018;21:388-93

How to cite this URL:
Jagdale S, Bhargava K, Bhosale S, Kumar T, Chawla M, Jagtap P. Comparative evaluation of coronal discoloration induced by two triple antibiotic revascularization protocols when used at varying depths of temporary sealing material at the end of varying time periods. J Conserv Dent [serial online] 2018 [cited 2022 Aug 14];21:388-93. Available from:

   Introduction Top

Revascularization of the necrotic pulp has become an alternative treatment option for the apexification procedure for immature permanent teeth. Revascularization permits thickening of root wall by mineralized tissue with continuing physiological root development.[1],[2] One of the most important steps of revascularization procedure is the disinfection of the root canal system. To effectively remove infection from the root canal system, several investigators used antibiotic paste.[2],[3],[4],[5] The traditional triple antibiotic paste (TAP) containing ciprofloxacin, metronidazole, and minocycline was developed. Several reports have confirmed the good antimicrobial properties of this mixture in infected root canal.[1],[6],[7] Furthermore, investigated TAP over various experimental periods and found it to be biocompatible. Out of these positive features, several case reports have proved that Minocycline causes visible crown discoloration.[8],[9],[10] Recently, antibiotics substitute to minocycline have been preferring for use in combination with ciprofloxacin and metronidazole, including amoxicillin, cefaclor, and doxycycline.[2],[4],[5],[11],[12]

When systemic antibiotics appear to be clinically effective as a companion in certain surgical and nonsurgical endodontic procedures, their administration is not without the probable risk of adverse systemic effects, such as allergic reactions, toxicity, and the development of resistant strains of microbes. In extension, the systemic administration of antibiotics count on patient acceptance with the dosing regimens followed by absorption through the gastrointestinal tract and distribution through the circulatory system to bring the drug to the infected site. Accordingly, the infected area requires a normal blood supply which is no longer the case for teeth with necrotic pulps and for teeth without pulp tissue. Hence, local application of antibiotics within the root canal system may be a more effective mode for delivering the drug.[13]

Murvindran has also advocated leaving of the triple antibiotic paste inside of the infected space for at least 3 weeks.[14] This was enough to not only disinfect the space but also to lead to a revascularization of the dead space.[15] Case studies have also reported the formation of immature root apices secondary to treatment with the triple antibiotic paste.

The third ingredient of the triple antibiotic paste was minocycline, and this gave the added benefit of being a collagen cross-linker.[16] However, the usage of minocycline led to the discoloration of the existing tooth structure.[17] This raised serious concerns as the usage of the triple antibiotic paste was maximum in the anterior teeth which suffered from childhood trauma most often, leading to the incomplete development of the root.[18]

This study was an effort to examine if the usage of the temporary restorative material at deeper depths would contravene the outcome of minocycline on the discoloration of teeth.

The rationale being that the lesser the number of tubules that were exposed to the effects of minocycline lesser would be the discoloration that was seen in the tooth structure.

Here, the readings for the discoloration were taken at the time intervals of 0, 3, and 6 weeks, which is the prescribed period, for which the TAP is to remain in the root canal.

It was evidenced that the color change was higher with minocycline and lesser with cefaclor and also that the color deepened with time.

   Materials and Methods Top

Forty extracted permanent maxillary central and lateral incisors were collected and chosen for the study. They were then divided into two groups and each group was then subdivided into two subgroups.

Storage of the samples

  • The apical 2–3 mm of the root was chopped off to leave behind 10–12 mm of root length from the cementoenamel junction (CEJ)
  • These trimmed roots were then stored in a formalin solution until the time processing of the samples.

Processing of the samples

  • The pulp was removed from the root canals, and then the teeth were stored overnight in 5% sodium hypochlorite (NaOCl), followed by washing under running water for 30 min
  • A Peeso reamer No-4 was used to enlarge the apical portion of the remaining root, in distribution to simulate unformed roots
  • The CEJ at this point was marked using permanent markers. The coronal part of the pulpal cavity was filled up to the marked CEJ using a temporary restorative material-Cavit™
  • Using a graduated probe and considering the CEJ as a starting point further marking was made on the tooth surface at 2 and 4 mm levels depending upon the group being processed
  • The temporary restorative material was then used to fill up the tooth to this marked point. The entire above procedure was done under an operating microscope
  • Equal portions of the three drugs were mixed with distilled water in a powder/liquid ratio of 3:1 to form a paste

    • TAP-1 – (metronidazole + ciprofloxacin + cefaclor)
    • TAP-2 – (metronidazole + ciprofloxacin + minocycline)

  • The apical openings were then sealed with Cavit-G and radiographs were taken to confirm void-free restorations. All the approved samples were then stored at 100% humidity in an incubator at 37°C for 3 weeks.

Tooth color assessment

  • Color measurements were recorded immediately after tooth preparation, on day 1, which was week 0, and then these readings were repeated at the end of the 3rd and the 6th week after placement of the medication
  • A standardized metal circular strip with a diameter of 5 mm was then placed on the labial surface of the crown 2 mm above the CEJ
  • Then, etching gel (37% phosphoric acid) was applied on the circumferential area of the ring for 30 s and washed off with water
  • The etched area was then outlined with oil paint to ensure that color measurement is carry out on the same region at every turn with a vertical angle.

Group A – two subgroups

  • TAP 1 – A mixture of – (metronidazole + ciprofloxacin + cefaclor) at 2 mm of the restorative material to be observed at the end of 0, 3 and 6 weeks
  • TAP 1 – A mixture of – (metronidazole + ciprofloxacin + cefaclor) at 4 mm of the restorative material to be observed at the end of 0, 3 and 6 weeks.

Group B – two subgroups

  • TAP 2 – A mixture of (metronidazole + ciprofloxacin + minocycline) at 2 mm of the restorative material to be observed at the end of 0, 3 and 6 weeks
  • TAP 2 – (metronidazole + ciprofloxacin + minocycline) mixture at 4 mm of the restorative material to be observed at the end of 0, 3 and 6 weeks.

Equal portions of the three drugs were mixed with distilled water in a powder/liquid ratio of 3:1 to form a uniformed paste.

  • Material was filled into each tooth as per grouping
  • Forty teeth samples were filled as per the above-mentioned groups and sealed using Cavit-G.

Evaluation and comparison of all groups and subgroups were done at the end of 0, 3, and 6 weeks using a photospectrometer.

   Discussion Top

Control of infection and complete asepsis of the root canal system is an objective that is sought after while doing root canal therapy. Even in the root canals that are not severely infected this becomes a challenge, and more so in canals which are severely afflicted with multimicrobial organisms. It has always been an uphill task to achieve gnotobiosis, even in ordinarily infected canals, and this task then becomes herculean when the clinician is faced with a weeping canal or a periapical lesion that exceeds 2 mm. The role of microorganisms in the development and sustain of pulp and periapical diseases has clearly been demonstrated in animal models and human studies. The development and process of endodontically generated periapical lesion is clearly associated with the presence of microorganisms in the root canal system. Bacteria in infected root canals and periradicular tissues are capable of invading and residing deeply within dentin and in cementum around the periapex. Endodontic therapy is advised at elimination of bacteria from the infected root canal and at the prevention of infection. The infection of the root canal system is considered to be a polymicrobial infection, consisting of both aerobic and anaerobic bacteria. Because of the difficulty of the root canal infection, it is doubtful that any single antibiotic could result in effective sterilization of the canal. More likely, a combination would be needed to address the diverse flora confront. The combination that appears to be most promising consists of metronidazole, ciprofloxacin, and minocycline, so that metronidazole was selected as the first choice of drug among antibacterial drugs. Even at a high percentage, it cannot kill all the bacteria, indicating the necessity for combination of other drugs. Propylene glycol can be used as a vehicle for this paste. The triple-antibiotics combination was first tested by Sato et al.[18] Teeth with immature root development, necrotic pulps, and apical periodontitis present multiple challenges for successful treatment.[19]

It is also clearly reveal in their in situ study that the mixture of ciprofloxacin, metronidazole, and minocycline goes through the dentine from the root canal and eradicated bacteria from the infected root dentine. This strongly proposed that infected root dentine can be sterilized by topical application of the drug combination to root canals in root canal treatment. Since the over large majority of bacteria in the deep layers of infected dentine of the root canal wall consist of obligate anaerobes (Ando and Hoshino 1990), metronidazole was selected as the first drug of choice among antibacterial drugs.

Typically, the sensitivity of bacteria to antibiotics is concluded as the minimum inhibitory concentration (MIC). Hoshino et al. suggested that the MIC method may not be suitable for calculating whether combinations of drugs can kill all the bacteria in such a flora. Hoshino et al. concluded that a combination of ciprofloxacin, metronidazole, and minocycline at a concentration of 25 g each per milliliter of paste was able to sterilize infected root dentin in vitro. Sato et al.[18] found that this combination at 50 g of each antibiotic per milliliter was sufficient to sterilize infected root dentin in situ.[18]

Interappointment intracanal medication has been clearly shown to contribute to favorable outcomes during treatment of endodontic infections. The need for intracanal medication is greater in those cases where bacteria are resistant to routine treatment, and where the therapy cannot be successfully completed due to the pain or continuing exudates. Sometimes endodontic conditions are treated over several appointments which may be extended over a long period. This permits various medicaments to be used depending on the status of the pulp, the periapical tissues, the hard dental tissues (such as cementum), and the condition of the apical foramen (i.e., “open,” or fully developed and unaffected by resorption). The minimum interappointment time interval should be not <14 days since inflammation takes at least 10–14 days to subside or heal, but longer periods are generally more prudent as most medicaments take 3–4 weeks to reach their maximum concentration within the peripheral dentine. In extension, if signs or symptoms are not subsiding, then a longer period of medication time or another medicament may be necessary.

Productive preparations in this group contain one or a combination of antibiotics, and sometimes other compounds such as corticosteroids. Antibiotics can be used as an adjunct to endodontic treatment in a number of ways – locally (i.e., intracanal), systemically, and prophylactically.

This latter study indicated that some medicaments should not be used in combination and that when two medicaments with strong antimicrobial activity are combined, there may be no additions or synergistic effects. A combination of antibiotics would also decrease the possibility of the development of resistant bacterial strains. Hoshino et al. concluded that a combination of ciprofloxacin, metronidazole, and minocycline, each at a concentration of 25 μg/ml (0.0025%) of paste, was able to disinfect infected root dentine in vitro. Sato et al. found that combination at 50 μg of each antibiotic/mL (0.005%) was sufficient to disinfect the infected root dentine.

Nevertheless, it is doubtful whether this concentration would be adequate in vivo, especially in immature teeth which present many challenges for their disinfection protocols, including the ability for periapically derived fluid to have a washing-out effect on the antibiotic paste through the open apical foramen. Portenier et al. show that dentine itself can have an inhibitory effect on the bactericidal activity of intracanal medicaments. So that, Windley et al. used metronidazole, ciprofloxacin, and minocycline in a thick paste at a concentration of 20 mg of each drug per mL (i.e.,2%) to move on these potential effects.

The most commonly used medicament is a combination of three antibiotics, referred to as a triple antibiotic paste (TAP). This combinations was first used by Sato et al. and included metronidazole, ciprofloxacin, and minocycline. This combination is available s Metronidazole is a nitroimidazole compound. It is selectively toxic to anaerobic microbes. Tetracyclines, which contain doxycycline and minocycline are primarily bacteriostatic, displays protein synthesis by binding to 30 s ribosomes in susceptible organisms. They exhibit broad spectrum of activity against Gram-positive and Gram-negative microorganisms. Minocycline is a semisynthetic derivative of tetracycline with a similar spectrum of antibacterial activity. Tetracycline shows collagenases and matrix metalloproteinases and is not cytotoxic. Ciprofloxacin is a synthetic fluoroquinolone with rapid bactericidal action. The bactericidal action probably results from digestion of DNA by exonucleases whose production is signaled by the damaged DNA. It reveals very powerful activity against Gram-negative bacteria but very limited activity against Gram-positive bacteria. Most of the anaerobic bacteria are resistant to ciprofloxacin. Hence, it is often combined with metronidazole in treating mixed infections.

Even though the triple antibiotic paste initially introduced by Hoshino et al. suppresses bacteria in the root canal, it had a disadvantage of causing esthetic problem leading to tooth discoloration. Kim et al. 2010 identified the discoloration was caused by minocycline in an in vitro study. Either an antibiotic alternate to minocycline is used, or it is omitted (Bi-Antibiotic paste). Trope et al. suggested that Arestin can be used as substitute for minocycline to reduce discoloration. Thomson A and Kahler B used alternative amoxicillin for minocycline in his case report to avoid discoloration. Other alternate measure is to eliminate or reduce discoloration through internal bleaching. Miller et al. performed internal bleaching to remove the cervical discoloration from the triple antibiotic paste 6 months later.[20]

The highest percentage of coronal discoloration occurred in the teeth filled with TAP with minocycline at all time intervals. The degree of discoloration increased with time and the perceptibility threshold from day 1 of the evaluation. Nosrat et al. used a TAP with minocycline for 4 weeks in immature central incisors. They noted moderate discoloration after 6 years. Lenherr et al. used a bovine tooth model to explore the discoloration potential of TAP with minocycline and other endodontic materials. They showed that TAP with minocycline-induced discoloration. Kim et al. tested three antibiotics (metronidazole, ciprofloxacin, and minocycline) and found that minocycline caused tooth discoloration. The results of the aforementioned studies confirm our results. The mechanisms by which TAP with minocycline impacts on coronal tooth discoloration may relate to the binding of minocycline to the calcium ions through chelation. Limited data are available in the literature concerning the tooth discoloration induced by antibiotic pastes, except for TAP with minocycline, which has been studied previously. The color of teeth after 3 weeks according to the groups: (a) control group, (b) calcium hydroxide, (c) double antibiotic paste (DAP), (d) TAP with minocycline, (e) with doxycycline, (f) with amoxicillin, and (G) with cefaclor, doxycycline, amoxicillin, or cefaclor also resulted in clinically visible crown discoloration. However, DAP and calcium hydroxide did not cause visible discoloration at any of the studied time points. However, there are no previous study data to compare the results of TAP with doxycycline, amoxicillin, or cefaclor and DAP. Lenherr et al. reported that the calcium hydroxide dressings did not show any discoloration or differ from the negative controls at any time point.

From the above-mentioned study, it is clear that the tooth needs to be protected against the deleterious effects of the discoloring agent. As it is the diffusion of the drug through the dentinal tubules that causes the discoloration, this study was undertaken with the objective of blocking this pathway using temporary restorative material. Furthermore, given that the pattern and diameters of the dentinal tubule vary at various levels, the depth (2 and 4 mm below the CEJ) to which the temporary was inserted was varied. A further testing component was added when the discoloration caused by the various agents was recorded at varying time intervals of 0, 3, and 6 weeks.

The values recorded at 0 weeks acted as the baseline values of each tooth. This thus standardized the actual color change that occurred in each tooth individually. The SS 5100H Spectrophotometer was used. (Manufacturer: Premier Colorscan Instruments Pvt. Ltd. Mumbai).

All the procedures, including the removal of pulp tissue, irrigation, placement of antibiotic pastes, and placement of cotton pellet and temporary filling material, were performed from the apical aspect to avoid disruption of the intact crown and to prevent coronal microleakage. The pulp tissue was removed, and the root canal was irrigated with 10 mL 5.25% NaOCl to remove any remaining pulp tissue; 10 mL 17% ethylenediaminetetraacetic acid was added for 2 min followed by 10 mL distilled water to simulate clinical irrigation. After the root canal space was dried using cotton, the pulp chamber was filled with temporary filling material (Cavit G 3M ESPE) from the pulp chamber ceiling up to 2 mm below the CEJ. A cotton pellet was placed loosely on the temporary filling material up to the facial CEJ. The specimens were then filled according to the group that they belonged to, thus effectively giving us 4 groups as follows:

The results obtained at the end of this study reiterated the fact that blocking of dentinal tubules by the application of temporary restorative material effectively decreases the color change in the coronal tooth structure. Furthermore, it was seen that the greater color change was seen when TAP 2 containing metronidazole, ciprofloxacin, and minocycline. This was in accordance with findings of Thomas (2014).

From this study, it is clear that the effect of the discoloring agent, in this case the various antibiotics is related, not only to the number of dentinal tubules exposed but also to the time of exposure. Hence, the longer that the antibiotic is in contact with the tooth structure, greater will be the amount of discoloration that occurs. Each tooth has a definite color before the manipulation, and this variable was factored out by taking a baseline reading for each sample before the sample is processed.

Furthermore, the area where the discoloration was measured was kept constant by marking off an area on the labial surface of each tooth. This further enhanced the standardization of the study design. These considerations reduced the variability induced by the condition of the tooth, the age of the patient, and the reason for extraction of that tooth.

Thus, in this study, the group that showed the least discoloration was the group containing metronidazole, ciprofloxacin, and cefaclor as opposed to the second TAP which contained metronidazole, ciprofloxacin, and minocycline. Furthermore, the color deepened at the end of 6 weeks. That is, there was a steady increase in the discoloration with the increase in time. This could have repercussions clinically when the TAP is used in anterior teeth. However, using the temporary restorative material to physically block the dentinal tubules showed a decline in the discoloration in both the groups with TAP 1 and TAP 2.

Further in roads could be made into the understanding of this discoloration if teeth were examined at more frequent intervals. Furthermore, other combinations of antimicrobials could be tested to check which one of them causes the least discoloration.

   Conclusion Top

  1. TAP 1 and TAP 2 both showed an increase in discoloration
  2. Greater discoloration was seen with TAP, containing minocycline
  3. There was a steady change in the discoloration from 0 to 6th week
  4. In both the groups, TAP 1 and TAP 2, there was a reduction in discoloration with increase thickness of the temporary restorative material.


I would like to thank Dr. Shalini for her guidance in the study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Shah N, Logani A, Bhaskar U, Aggarwal V. Efficacy of revascularization to induce apexification/apexogensis in infected, nonvital, immature teeth: A pilot clinical study. J Endod 2008;34:919-25.  Back to cited text no. 1
Namour M, Theys S. Pulp revascularization of immature permanent teeth: A review of the literature and a proposal of a new clinical protocol. ScientificWorldJournal 2014;2014:737503.  Back to cited text no. 2
Vijayaraghavan R, Mathian VM, Sundaram AM, Karunakaran R, Vinodh S. Triple antibiotic paste in root canal therapy. J Pharm Bioallied Sci 2012;4:S230-3.  Back to cited text no. 3
Bansal R, Jain A. Overview on the current antibiotic containing agents used in endodontics. N Am J Med Sci 2014;6:351-8.  Back to cited text no. 4
Pedrosa MT, Yuri J. Pulp revascularization: An alternative treatment to the apexification of immature teeth. RGO Rev Gaúch Odontol Porto Alegre 2004;62:401-10.  Back to cited text no. 5
Mohammadi Z, Shalavi S, Yazdizadeh M. Antimicrobial activity of calcium hydroxide in endodontics: A review. Chonnam Med J 2012;48:133-40.  Back to cited text no. 6
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Wang DD, Englot DJ, Garcia PA, Lawton MT, Young WL. Minocycline- and tetracycline-class antibiotics are protective against partial seizures in vivo. Epilepsy Behav 2012;24:314-8.  Back to cited text no. 16
Antonini LG, Luder HU. Discoloration of teeth from tetracyclines – Even today? Schweiz Monatsschr Zahnmed 2011;121:414-31.  Back to cited text no. 17
Duggal M, Tong HJ, Al-Ansary M, Twati W, Day PF, Nazzal H, et al. Interventions for the endodontic management of non-vital traumatised immature permanent anterior teeth in children and adolescents: A systematic review of the evidence and guidelines of the European Academy of Paediatric Dentistry. Eur Arch Paediatr Dent 2017;18:139-51.  Back to cited text no. 18
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Correspondence Address:
Dr. Karan Bhargava
Senior Lecturer, Dr. D. Y. Patil Dental College & Hospital, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharastra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JCD.JCD_117_17

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