Evaluation of Cytotoxicity of Premixed Triple Antibiotic Paste on Stem Cells of Apical Papilla Using MTT Assay: An In Vitro Study

Abstract

Background

Endodontic regenerative procedures are biologically based and intend to replace damaged dental tissues. This includes the restoration of the dentin-pulp complex through the use of stem cells. Root canal medications, particularly antibiotics, have shown significance in obtaining excellent therapeutic outcomes. However, high concentrations of some materials, like calcium hydroxide [Ca(OH)₂] and mineral trioxide aggregate (MTA), can be cytotoxic, which necessitates a search for less toxic alternatives.

Aim

To evaluate the cytotoxic effects of premixed triple antibiotic paste (TAP) on stem cells of apical papilla (SCAPs) using methyl thiazolyl tetrazolium (MTT) assay.

Materials and methods

Stem cells of apical papilla were isolated from the dental pulp tissue of extracted premolars and characterized using flow cytometry. The isolated cells were divided into four groups: Groups I, II, and III were treated with TAP at concentrations of 10, 25, and 50 μg/mL, respectively, while group IV served as the untreated control. The MTT assay was performed according to the manufacturer's protocol to assess cell viability. Evaluations were carried out at 1, 3, and 7 days following exposure to the TAP concentrations.

Results

Cell viability was highest in groups treated with 10 and 25 μg/mL TAP, indicating minimal cytotoxicity across all evaluated time points—1, 3, and 7 days. In contrast, the group exposed to 50 μg/mL TAP demonstrated significantly reduced cell viability, indicating a dose-dependent increase in cytotoxicity.

Conclusion

This study concludes that TAP at concentrations of 10 and 25 μg/mL demonstrates higher cell viability and lower cytotoxicity on SCAPs across 1, 3, and 7 days of evaluation. In contrast, a concentration of 50 μg/mL exhibited the most pronounced cytotoxic effects. While these findings suggest that lower concentrations of TAP may be safer for regenerative applications, further in vivo studies and preclinical animal trials are warranted to confirm biocompatibility and clinical feasibility prior to translation into human use.

How to cite this article

Saravanan E, Ravindran V. Evaluation of Cytotoxicity of Premixed Triple Antibiotic Paste on Stem Cells of Apical Papilla Using MTT Assay: An In Vitro Study. Int J Clin Pediatr Dent 2025;18(9):1082–1086.

Introduction

Endodontic regenerative techniques are biologically based procedures designed to restore damaged tissues, such as dentin, root structure, and cells that make up the dentin-pulp complex cells.¹ The ultimate objective is to increase tooth longevity and function by replacing the infected or dead pulp tissue with the pulp-dentin complex cells.² Pediatric endodontic therapy is used when a deciduous tooth's pulp sustains permanent damage in order to preserve the tooth's function up until the scheduled exfoliation phase. As an adjuvant to therapeutic treatment, root canal medications—especially antibiotics—have continuously shown their critical importance and primary function in achieving excellent outcomes.³

Advanced biomedical engineering-focused three-dimensional in vitro tissue culture systems offer valuable frameworks for replicating and investigating the molecular and cellular interplay governing the development and regeneration of teeth. This includes the mechanisms that oversee dental stem cell (DSC) mediated dental tissue homeostasis, regeneration, and repair.⁴ Research findings indicated that calcium hydroxide [Ca(OH)₂] had the capacity to stimulate cell growth and improve the survival of stem cells, but this effect was observed solely at lower concentrations of the compound.⁵ As noted by Khoshkhounejad et al.,⁶ the utilization of a Ca(OH)₂ paste resulted in a significant decrease in cell viability due to pronounced cytotoxicity associated with the highly concentrated, pasty consistency of Ca(OH)₂. Importantly, compounds based on MTA, Biodentine, and Ca(OH)₂ can be very effective antibacterial agents in the field of endodontics. However, it is worth noting that their effectiveness in eradicating bacteria is dependent on the application of high concentrations, which may inadvertently compromise the survival of cells.⁷

The American Association of Endodontists recommends intracanal placement of antibiotic pastes, which has been widely adopted in regenerative endodontic procedures, but the usage of triple antibiotic paste (TAP) has also been associated with adverse clinical consequences.^8,9 These include disruptions in the release of dentin growth factors, hindrance of vascularization largely due to minocycline presence, and the notable presence of high toxicity.^10,11

Using a combination of three antibiotics—metronidazole, ciprofloxacin, and minocycline—lesion sterilization and tissue repair therapy (LSTR) or noninstrumentation endodontic treatment (NIET) is done, they are innovative biological strategies for managing carious lesions, whether there is involvement or no involvement of the pulp or periapical areas.¹² This mixture is effectively active against a variety of bacteria, whether they are gram positive or gram negative, obligatory or facultative, and promotes the healing of the wound.

It has also been documented that high concentration of TAP exerts cytotoxic effects on the primary pulp stem cells of deciduous teeth.¹ The use of TAP in endodontics has sparked debate due to concerns regarding its potential side effects, such as tooth discoloration and cytotoxicity towards dental pulp cells. Some practitioners opt to mix antibiotics at the point of use to precisely regulate the concentration and ensure compatibility with other materials used during the procedure.

Methyl thiazolyl tetrazolium (MTT) assay is considered as gold standard for testing cytotoxicity of the materials.¹³ The principle of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is based on the conversion of a yellow tetrazolium salt, MTT, into a purple formazan crystal by mitochondrial enzymes within metabolically active cells.¹⁴

Thus, this study's primary goal was to evaluate the cytotoxicity of a premixed triple antibiotic formulation on human apical dental pulp stem cells using MTT assay.

Materials and Methods

Experimental Design

Sample Size

The sample size was calculated from a similar study by Rafatjou et al.¹ The G*Power software version 3.0 for power analysis indicated that a total of 12 teeth (corresponding to achieve 80% power with 95% confidence interval) were required.

Ethical Approval and Consent

The study was conducted at a private dental institution in Chennai and received ethical clearance from the Institutional Review Board (Approval Number: SRB/SDC/PEDO-2203/23/087). Following the patients' informed consent, 12 healthy immature first premolars with an open apex were chosen to extract SCAPs from six healthy patients in 8–10-year age range who had been referred due to orthodontic reasons to the Pediatric Dentistry Department and had no prior history of systemic diseases.

Extraction of Dental Pulp

The dental pulp was obtained from premolars, which were indicated for extraction due to orthodontic reasons. The patient received comprehensive oral health education and prophylaxis 2–5 days prior to tooth extraction. On the day of the extraction, the patient also received teeth prophylaxis. Patients washed their mouths with 0.2% Chlorhexidine mouthwash for 30 seconds both before and after receiving an anesthetic injection. Dental pulp was extracted, and SCAPs were characterized through flow cytometry.

Cell Culture

The extracted teeth were promptly immersed in warm, sterile phosphate-buffered saline (PBS) (Gibco, USA; Idehzist), supplemented with 1% penicillin. Using sterile dental tweezers, the apical papilla tissue was carefully isolated and transferred into an enzymatic digestion solution containing 3 mg/mL collagenase type I (Sigma, Germany) and 1% Penicillin–Streptomycin. The resulting cell suspension was cultured in alpha-modified Eagle's medium (α-MEM) (Gibco, USA; Idehzist), supplemented with 20% fetal bovine serum (FBS) (Sigma-Aldrich, Germany) and 1% Penicillin–Streptomycin to prevent microbial contamination, with penicillin primarily targeting gram-positive bacteria and streptomycin effective against gram-negative bacteria. The culture medium was replaced every 48 hours, and to prevent fungal growth, Fungisome (Amphotericin B) was added at a concentration of 2 μg/L.

Flow Cytometric Surface Marker Expression Analysis in HDPSCs

Flow cytometry was employed to confirm the mesenchymal stem cell (MSC) identity of the isolated cell population and to eliminate the presence of non-MSCs. The analysis was carried out using a Cytomics FC500 flow cytometer (Beckman Coulter, USA) operated with CXP software version 2.2. For surface marker profiling, adherent cells were enzymatically detached and resuspended to achieve a final concentration of 1 × 10⁶ cells/mL. A total of 1 × 10⁶ cells were then incubated with 10 µL of fluorochrome-conjugated monoclonal antibodies specific for CD90 and CD105 (to confirm mesenchymal origin) and CD34 (to exclude hematopoietic lineage), all obtained from Beckman Coulter, USA. The staining procedure was conducted at 4°C in darkness, with matched isotype controls used to assess nonspecific binding. Following a 20-minute incubation, 2 mL of PBS supplemented with 2% fetal bovine serum (FBS) was added to each sample tube. The samples were centrifuged at 2,500 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and the resulting cell pellet was resuspended in 500 µL of PBS containing 2% FBS for flow cytometric acquisition.

Preparation of Test Materials (P-TAP)

The antibiotics used for preparing TAP were Metronidazole (500 mg) (Flagyl, Abbott, India) tablets, Minocycline (Minoz, Sun Pharmaceutical Industries, India) (100 mg) tablets, and Ciprofloxacin (500 mg) (Ciplox, Cipla, India) tablets. The antibiotics were triturated separately using mortar and pestle and made into fine powder. The antibiotics were taken in a ratio of 1:1:1 (Ciprofloxacin:Metronidazole:Minocycline). Each medication was taken in a separate centrifuge tube, and 5 mL of distilled water was added for each tube and shaken well. Then, all three medications were poured into a single centrifuge tube and kept in a shaker overnight for uniform mixing. Then 10 gm of empty gel was added to the triple antibiotic solution and mixed well till the desired flowable consistency was obtained. Gelatin, a protein known for forming solid gels in water-based solutions, is widely utilized for its strong gelling capabilities. Its utility extends beyond the food industry, finding significant application in the biotechnology sector, particularly as a protective coating for pharmaceutical compounds.

For all the groups antibiotics are prepared in the same way, only the concentration of each group is changed by adding PBS.

• Group I: Stem cells of apical papilla (SCAPs) were exposed to the concentration of 10 μg/mL for a time period of 1, 3, and 7 days.

• Group II: Stem cells of apical papilla were exposed to the concentration of 25 μg/mL for a time period of 1, 3, and 7 days.

• Group III: Stem cells of apical papilla were exposed to the concentration of 50 μg/mL for a time period of 1, 3, and 7 days.

• Group IV: Control group contains untreated cells.

Cell Viability Assay

The cytotoxic potential of the test materials was assessed using the MTT assay kit (VACSERA, Giza, Egypt), adhering strictly to the protocol provided by the manufacturer. Cell cultures were maintained in Eagle's minimum essential medium (MEM-E), supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acids, 100 IU/mL Penicillin, and 100 µg/mL Streptomycin dissolved in 0.9% Sodium chloride. Cultures were maintained under standard conditions in a humidified incubator at 37°C with 5% CO₂ and 95% air, allowing the formation of a confluent monolayer. All culture reagents were sourced from Sigma-Aldrich (St. Louis, MO, USA).

Upon reaching confluence, the culture medium was removed, and cells were detached by exposing them to 0.25% Trypsin solution (Sigma-Aldrich, USA) for approximately 2–3 minutes. Trypsinization was monitored and terminated once complete cellular dissociation was achieved. The cells were subsequently collected and preserved under appropriate conditions for subsequent experimental use. The suspended cells were counted and dispersed in 96-well tissue culture dishes using a hemocytometer.¹⁵

The MTT assay, which measures cytotoxicity, involved dissolving 50 mg of MTT powder in 10 mL of PBS and filtering the solution through sterile filters with a 0.22 µm pore size. Using a 1:1:1 weight ratio, metronidazole, ciprofloxacin, and minocycline were combined to create TAP. The group maintained in standard culture medium without any treatment served as the negative control. Cell viability in this group was used as a baseline for comparison with the treated groups.²

Statistical Analysis

The experiments were done in triplicate, and the results were expressed as mean and standard deviation. The percentage of viable HDPSCs in the test groups, relative to the untreated control, was analyzed using one-way analysis of variance (ANOVA) and intergroup and intragroup comparison was assessed using repeated measures ANOVA in the Statistical Package for Social Sciences (SPSS) software version 22 (IBM SPSS Statistics for Windows, ver. 22.0, Armonk, New York: IBM Corp.). The difference is considered significant if the p value is <0.05.

Results

The results of this study reveal significant variations in percentage viability across different time points (at day 1, 3, and 7) and different concentration levels. At day 1, the mean percentage viability for group I was 92.91% with a standard deviation (SD) of 0.08%, while group II had a mean viability of 83.32% (SD = 0.28%), and group III was 69.80% (SD = 0.26%). In contrast, the group IV had a mean percentage viability of 99.60% (SD = 0.53%) at day 1.

As time progressed, the mean percentage viabilities for the groups I, II, and III decreased significantly at day 3 and day 7, indicating a time-dependent effect. The p values, which assess the significance of these differences, were all less than 0.05, with the lowest at 0.005, indicating highly significant differences between the groups.

In contrast, the group IV showed no significant changes in percentage viability over time, with p-values consistently above 0.05. These findings suggest that the percentage viability of groups I, II, and III decreases significantly over the course of 7 days, while the control group remains relatively stable (Table 1).

Table 1:

Evaluation of cell viability among the material tested in present study at different time periods

	1st day		3rd day		7th day		p-value
	Mean	SD	Mean	SD	Mean	SD
Group I	92.91	0.08	85.58	0.62	82.64	0.74	0.033b
Group II	83.32	0.28	72.13	0.38	68.11	0.27	0.012b
Group III	69.80	0.26	49.06	0.12	45.11	0.11	0.005b
Group IV	99.60	0.53	97.07	0.06	95.47	0.42	0.175b
p-value	0.00a		0.00a		0.00a

^aStatistically significant with p < 0.05 based on one-way ANOVA; ^bStatistically significant with p < 0.05 based on repeated measures ANOVA

Discussion

This study assessed the cytotoxic effects and cell viability of TAP at different concentrations (10, 25, and 50 μg/mL) over a period of 1, 3, and 7 days. The results indicate that lower concentrations of TAP (10 and 25 μg/mL) maintain better cell health and exhibit less toxicity compared to the higher concentration of 50 μg/mL, which shows significantly greater cytotoxic effects. These findings suggest that minimizing the concentration of TAP could optimize endodontic treatment by effectively disinfecting the root canal while preserving surrounding cellular health.

The imperative for successful regeneration of the dentin-pulp complex lies in the establishment of a microbe-free environment using medicaments that have minimal toxic effects on stem cells.¹⁶ Efforts to identify an appropriate medicament are ongoing, with studies investigating various dilutions to assess both cytotoxicity and antibacterial effectiveness.¹⁷ In contrast to prior research, this study specifically aimed to evaluate the cytotoxic impact of various medicaments on stem cells.

The normal microbial flora will only be suppressed by a nonspecific antibiotic therapy, which will provide a chance for the persistent, pathogenic residual bacteria to repopulate the canal area. Therefore, a combination of antibiotics is required to completely eradicate the canal pathogens and destroy the resistance of the aggressive bacteria.¹⁸ Three antibacterial drugs with distinct action spectra and separate methods of action—metronidazole, minocycline, and ciprofloxacin—combine to form the paste. Metronidazole is a broad-spectrum antibiotic that binds to DNA and breaks its helix, making it effective against anaerobic bacteria. A semisynthetic tetracycline and minocycline prevent the creation of new proteins. Moreover, it has a wide range of effectiveness against both gram-negative and gram-positive bacteria. A fluoroquinolone called ciprofloxacin prevents bacteria's DNA gyrase from functioning. Although it is not as effective against gram-positive bacteria, it nevertheless demonstrates strong bactericidal action against gram-negative bacteria. When these medications are used together, the ecology of different bacteria found in root canals is successfully eradicated, and antibiotic resistance is prevented.¹⁹

Due to concerns regarding the safety of stem cells, there is a growing inclination to utilize lower concentrations of intracanal medicaments. Recent studies have consistently demonstrated that reducing the concentration of medicaments significantly lowers their cytotoxic effects. However, it is essential to acknowledge that disparities in methodologies and materials across these studies might hinder direct comparisons with the current study.²⁰

In a study conducted by Khoshkhounejad et al., TAP was identified as the most benign drug for SCAPs, whereas Augmentin demonstrated certain detrimental effects.⁶ Concerns regarding the safety of stem cells have prompted a shift toward utilizing lower concentrations of intracanal medications. Previous studies have shown that the viability of SCAPs decreases in a concentration and time-dependent manner when exposed to double antibiotic paste (DAP) and TAP. Notably, significant cytotoxic effects were observed at a concentration of 100 mg/mL of DAP after 24 hours, and at both 10 and 100 mg/mL concentrations following 48 and 72 hours of exposure, compared to the control group. Additionally, TAP considerably reduced SCAPs' cell viability in comparison to control across all treatment doses and time periods.²

Another study concluded that TAP and mTAP had comparable cytotoxic effects on SCAPs at concentrations of 100 μg/mL over the studied time interval. This study also observed enhanced SCAP cell proliferation when treated with low-dose binary antibiotic combinations at concentrations of 25 and 50 μg/mL. These results could indicate that antibiotics behave similarly at lower doses while acting differently at greater quantities.¹ Another study demonstrated that calcium hydroxide (CH) promotes cellular proliferation and enhances early in vitro mineralization, whereas modified triple antibiotic paste (mTAP) exhibited higher cytotoxicity and reduced the differentiation capacity of apical papilla cells (APCs) in vitro.²¹ In contrast, the present study demonstrated that TAP at concentrations of 10 and 25 μg/mL exhibited minimal cytotoxicity across all evaluated time intervals—1, 3, and 7 days.

The use of low antibiotic doses to create antibiotic-releasing scaffolds has been explored in previous studies.²² Considering the advantages of sustained drug release from such scaffolds, it is essential to evaluate the cytotoxic effects of low-dose antibiotics on different stem cell populations, along with their antimicrobial efficacy against a range of bacterial strains. This research is vital as it may contribute to the development of scaffold materials.

The study's results highlight the importance of dose-optimized TAP for enhancing dental pulp cell viability, crucial for regenerative endodontic procedures. By using TAP at lower concentrations (10 and 25 μg/mL), practitioners can effectively disinfect the root canal while minimizing cytotoxicity, promoting pulp-dentin complex regeneration. This finding is pivotal for dose optimization, ensuring antimicrobial efficacy without compromising biocompatibility, essential for successful pulp therapy and endodontic regeneration, and underscores the significance of preserving tooth vitality and promoting tissue repair, especially in developing teeth.

The current study has assessed the cytotoxic properties of TAP to SCAPs, which are essential for tissue regeneration. The study has certain limitations that need to be acknowledged. Firstly, the findings are based on an in vitro study setup, and thus, their direct clinical applicability should be interpreted with caution and a smaller sample size was used in the current study. This study involved only a shorter period of follow-up. To establish clinical relevance and applicability, animal studies should be designed and conducted to evaluate the regenerative properties before considering the initiation of human trials.

Conclusion

This study concludes that TAP at concentrations of 10 and 25 μg/mL shows better cell viability and less cytotoxic effects whereas concentration of 50 μg/mL shows the highest cytotoxic effects at 1, 3, and 7 days.

Orcid

Elanthendral Saravanan https://orcid.org/0009-0002-0779-7764

Vignesh Ravindran https://orcid.org/0000-0002-7534-3636