Cytotoxicity of chitosan-loaded triple antibiotic paste versus conventional triple antibiotic paste on dental pulp stem cells: An in vitro study

Abstract

Background:

Triple antibiotic paste (TAP) is a commonly used intracanal medicament in regenerative endodontics. However, its cytotoxic effects on dental pulp stem cells (DPSCs) remain a concern. Incorporating chitosan into TAP may enhance its biocompatibility while retaining antimicrobial properties.

Aim:

The aim of this study was to evaluate and compare the cytotoxicity of chitosan-loaded TAP (Ch-TAP) with conventional TAP on DPSCs at varying concentrations and time intervals.

Materials and Methods:

DPSCs were exposed to Ch-TAP and TAP at concentrations of 0.1–100. Cell viability was assessed at 1–4 weeks using an MTT assay. The results were statistically analyzed using one-way ANOVA and post hoc Tukey’s test to identify differences in cytotoxic effects between groups.

Results:

Ch-TAP demonstrated superior cell viability at all concentrations compared to TAP. The highest cell viability was observed with Ch-TAP at 0.1 mg/mL, maintaining 96% viability at 1 week and 85% at 4 weeks. Higher concentrations of both pastes exhibited a dose dependent decrease in cell viability, with Ch-TAP consistently showing less cytotoxicity. TAP at 100 mg/mL exhibited the highest cytotoxicity, reducing viability to 20% by the 4^th week.

Conclusion:

Ch-TAP showed significantly lower cytotoxicity and better biocompatibility with DPSCs compared to conventional TAP, particularly at lower concentrations.

INTRODUCTION

Regenerative endodontics has rapidly evolved, shifting the focus from conventional root canal treatment, which primarily aims to disinfect and seal the root canal system, to procedures that encourage biological healing and tissue regeneration within the pulp–dentin complex. Disinfection of the root canal, especially where the microbial load is high, is crucial.

The triple antibiotic paste (TAP) is well established and documented for its broad-spectrum antimicrobial efficacy against endodontic pathogens.[1] TAP is well regarded for its ability to eradicate difficult-to-reach bacteria; however, concerns about its cytotoxicity and potential adverse effects on host cells.[2] Dental pulp stem cells (DPSCs) play a key role in pulp regeneration, dentin repair, and immune response modulation.[3] Unfortunately, conventional TAP, while effective in microbial suppression, has been shown to exert cytotoxic effects on DPSCs and other cells within the pulp, potentially limiting regenerative outcomes.[2] The presence of minocycline has been associated with the staining of dentin as well as cytotoxic effects, prompting efforts to replace or modify TAP components to improve biocompatibility.[4]

Chitosan is a natural polymer derived from the deacetylation of chitin found in crustacean shells and has shown great promise in biomaterial research due to its biodegradability, biocompatibility, and antimicrobial properties.[5,6] Chitosan not only exhibits intrinsic antibacterial effects but also serves as an effective carrier for drug delivery, potentially enhancing the stability and controlled release of antibiotics in endodontic applications.[7,8] In addition, chitosan has been shown to promote cellular proliferation and differentiation, which could benefit the regenerative process in endodontic treatments.[9] Methylthiazol tetrazolium bromide is a colorimetric assay that measures cell viability based on mitochondrial activity used to assess cytotoxic effects.

This study aims to compare the effects of chitosan-loaded TAP (Ch-TAP) versus TAP on DPSC using 3-(4,5-diethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different concentrations and provide the biocompatibility of these formulations.

MATERIALS AND METHODS

The study is approved by the Institutional Ethical Committee of Maharashtra Cosmopolitan Education Society under the approval number MCES/EC/531-A/2023.

Preparation of antibiotic pastes

Conventional TAP was prepared by mixing metronidazole, ciprofloxacin, and clindamycin powder. The mixture was dissolved in propylene glycol to prepare the combinations with final concentrations of 0.1 mg/mL, 1 mg/mL, 10 mg/mL, and 100 mg/mL. Chitosan powder (Nanoshel) was dissolved in 1% acetic acid, stirred for 24 h, and combined with metronidazole, ciprofloxacin, and clindamycin to form a paste.

Culture of dental pulp stem cells

DPSCs were isolated from freshly extracted the third molars under ethical approval, using informed consent from patients. The teeth were decontaminated with 2% chlorhexidine solution, and the pulp tissue was aseptically removed, minced, and enzymatically digested using 3 mg/mL collagenase type I and 4 mg/mL dispase for 30 min at 37°C. DPSCs in passages 2–5 were used for experiments. DPSCs were verified under microscopic [Figure 1a].

Figure 1.

Workflow of cytotoxicity testing using MTT assay, (a) Bright field microscopic image of passage 2 healthy human dental pulp stems, (b) Preparation of triple antibiotic paste (TAP), (c) Loading of TAP in syringe, (d) Preparation of chitosan-loaded TAP, (e) Loading of Ch-TAP in syringe, (f) 96-well plate 3-(4,5-diethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay plate showing the tested groups (c-group 9, 1-group 1, and 5-group 5 showing dark purple color indicating high cell viability; 2-group 2, 6-group 6, and 3-group 3 showing medium purple color indicating moderate cell viability; and 7-group 7, 4-group 4, and 8-group 8 showing light purple color indicating low cell viability)

Preparation of cell treatments

Each paste concentration was diluted in culture media and incubated for 24 h at 37°C [Figure 1b-e]. The extracts were filtered through a 0.22 μm membrane to remove particles. All extracts were prepared fresh before each time point evaluation.

Cytotoxicity testing

DPSCs were seeded in 96-well plates at a density of 5 × 10³ cells/well and incubated overnight for attachment. The next day, cells were treated with 100 μL of corresponding TAP or Chitosan loaded triple antibiotic paste (CL-TAP) extract for each concentration and the control group. After exposure periods of 1–4 weeks, methyl thiazolyl tetrazolium reagent (10 μL of 5 mg/mL) was added to each well, followed by a 4-h incubation at 37°C to allow formazan crystal formation in metabolically active cells [Figure 1f]. After which, 100 μL of dimethyl sulfoxide was added to dissolve the formazan crystals.

The study was as follows:

• Groups 1: Chitosan-loaded TAP at a concentration of 0.1 mg/mL

• Group 2: Chitosan-loaded TAP at a concentration of 1 mg/mL

• Group 3: Chitosan-loaded TAP at a concentration of 10 mg/mL

• Group 4: Chitosan-loaded TAP at a concentration of 100 mg/mL

• Groups 5: Conventional TAP at a concentration of 0.1 mg/mL

• Group 6: Conventional TAP at a concentration 1 mg/mL

• Group 7: Conventional TAP at concentration of 10 mg/mL

• Group 8: Conventional TAP at concentration of 100 mg/mL

• Group 9: Control group (untreated DPSCs in culture media).

Absorbance measurement and data analysis

Absorbance was calculated at 570 nm with a microplate reader. Cell viability was expressed as a percentage in comparison to the control group that received no treatment. Statistical analysis was done using a one-way ANOVA to assess concentration- and time-dependent effects and Tukey’s post hoc test for intergroup comparisons.

RESULTS

Effect of chitosan-loaded triple antibiotic paste on dental pulp stem cell viability

A dose-dependent reduction in cell viability was observed with increasing concentrations of Ch-TAP. The decrease was statistically significant across all time points [P < 0.001, Table 1]. Group 1 exhibited the highest viability at 1 week, which gradually declined by week 4. This trend continued across Groups 2 and 3, with Group 4 showing the lowest viability [Graph 1].

Table 1.

Overall intergroup comparison of average absorbance between the study groups using one-way ANOVA F-test

Groups	1 week, mean (SD)	2 weeks, mean (SD)	3 weeks, mean (SD)	4 weeks, mean (SD)
Group 1	87.8	88.4	81.6	74.8
Group 2	83	79.8	77	70
Group 3	79.4	77.2	72.2	63.8
Group 4	67.6	68.6	62.8	56.6
Group 5	80.4	78.8	72.8	69.4
Group 6	70.4	67	59.8	52.8
Group 7	52.4	51.8	44.4	38.7
Group 8	29.8	28.6	24.6	24.6
Group 9	100	100	100	100
One-way ANOVA F-test	790.22	646.72	528.70	369.81
P, significance	<0.001**	<0.001**	<0.001**	<0.001**

**P<0.001 – highly significant difference. SD: Standard deviation

Graph 1.

Cytotoxicity comparison of different concentrations of Chitosan loaded triple antibiotic paste (CL-Tap) with the control group on dental pulp stem cell viability at various time periods. C-TAP: Chitosan loaded triple antibiotic paste

Effect of conventional triple antibiotic paste on dental pulp stem cell viability

Similarly, TAP demonstrated a concentration dependent decrease in DPSC viability, with all differences being statistically significant at every time interval [P < 0.001, Table 1]. Group 5 maintained the highest cell viability at 1 week, followed by a gradual decrease at 4 weeks in Groups 6 and 7. Group 8 exhibited the lowest cell viability [Graph 2].

Graph 2.

Cytotoxicity comparison of different triple antibiotic paste concentrations with the control group on dental pulp stem cell viability at different time intervals. TAP: Triple antibiotic paste

Pairwise comparison (Tukey’s post hoc test) Group 1 showed a highly significant difference when compared with all other groups at 1 and 2 weeks (P < 0.001) and a significant difference with Groups 2, 3, and 5 at 4 weeks. No significant difference was noted between Groups 1 and 2 at 4 weeks. Group 2 showed significant differences with all groups except Group 3 at 1 week. Groups 3 and 4 differed significantly from Groups 4, 6, 7, and 8; however, no significant difference was observed between Groups 3 and 5 at 1 and 2 weeks.

DISCUSSION

Regenerative endodontics is gaining increasing significance as it shifts the paradigm significance from traditional root canal treatment toward biologically based therapies aimed at restoring the natural vitality and function of the tooth. The TAP is a widely used intracanal medicament in regenerative endodontics.[10] It has been extensively studied in both in vitro models and clinical trials. However, despite its effectiveness, its use in regenerative endodontics raises safety concerns, particularly related to its concentration. Ruparel et al. demonstrated that TAP at concentrations typically used in clinical settings significantly reduced cell viability, suggesting the need for dilution to safer levels.[11]

Chitosan, a natural biopolymer with inherent antibacterial properties, has been shown to reduce the cytotoxic effects.[5,8,9] The cytotoxic effects of TAP and calcium hydroxide have been reported by various studies.[2,11,12,13] The efficacy of combining chitosan with TAP against Enterococcus faecalis has been reported by Shaik et al.,[14] Hegde et al.,[5] and Swaroop et al.;[15] however, a comparison of traditional TAP versus Ch-TAP on cell viability, particularly concerning stem cells, is not been reported so far. To our knowledge, this is the first study comparing the cytotoxicity of Ch-TAP to conventional TAP on DPSCs at different concentrations and time intervals.

There are numerous methods used to assess the cell viability. In this study, yellow tetrazolium (MTT) was used considering the cost-effectiveness and accuracy of results.[16] This method allows easy and accurate differentiation of viable and nonviable cells. The findings of our study showed that Ch-TAP demonstrated significantly higher cell viability compared to TAP, particularly at lower concentrations, 0.1 mg/mL showing the highest viability (96%) at 1 week and a gradual decline over time. Our findings are consistent with the results reported by Jamshidi et al., who evaluated their study and showed that lowering the concentration of TAP significantly improved cell viability. Other studies have also highlighted the cytotoxic effects of high-concentration antibiotic pastes on stem cells, emphasizing the need for alternative formulations.[11,12,13,17] Ch-TAP at 0.1 mg/mL exhibited the highest cell viability across all groups, maintaining 96% at 1 week and decreasing to 85% by 4 weeks. Even at higher concentrations (1 mg/mL and 10 mg/mL), Ch-TAP demonstrated better viability compared to TAP. The superior cytocompatibility of Ch-TAP can be attributed to the bioactive properties of chitosan, which has been shown to enhance cell proliferation and reduce cytotoxicity by modulating drug release. This finding aligns with reports that chitosan acts as a biocompatible drug carrier, promoting tissue regeneration and minimizing cellular toxicity.[18]

Conventional TAP showed lower cell viability at all concentrations compared to Ch-TAP. At 0.1 mg/mL, TAP demonstrated a viability of 90% at 1 week, decreasing to 80% at 4 weeks. Higher concentrations, particularly 10 mg/mL and 100 mg/mL, resulted in significantly reduced cell counts, with the lowest viability observed at 100 mg/mL (20% at 4 weeks). Both Ch-TAP and TAP exhibited a dose dependent decrease in cell viability. While Ch-TAP at higher concentrations (100 mg/mL) showed a significant decline in cell viability (30% at 4 weeks), it was still less cytotoxic than TAP, which demonstrated only 20% viability at the same concentration and time point. The results of this study suggest that Ch-TAP, particularly at lower concentrations, could serve as a safer alternative to conventional TAP in regenerative endodontic procedures. The superior cytocompatibility of Ch-TAP aligns with findings from other studies, which emphasized the potential of chitosan-based materials to promote tissue healing and regeneration.[19,20,21,22] These findings are significant in addressing the challenges of balancing antibacterial efficacy with cytocompatibility, a major consideration in regenerative endodontics.

While the in vitro findings provide valuable insights, further studies are required to validate these results in vivo. Long-term evaluations of tissue regeneration outcomes and antimicrobial efficacy are also needed. Moreover, the molecular mechanisms underlying the reduced cytotoxicity of Ch-TAP should be explored, particularly focusing on the role of chitosan in modulating drug release and cellular interactions.

CONCLUSION

This study demonstrates that Ch-TAP is a promising alternative to conventional TAP in regenerative endodontics due to its significantly lower cytotoxicity and higher cell viability, especially at lower concentrations. The incorporation of chitosan not only enhances the biocompatibility of the paste but also provides a controlled release of antibiotics, minimizing their adverse effects on DPSCs. These findings emphasize the potential of Ch-TAP in promoting favorable outcomes in regenerative procedures. However, further in vivo studies and long-term evaluations are essential to confirm its clinical applicability and efficacy.

Disclosing statement

This in vitro cytotoxicity study is a part of PhD clinical trial. The findings here are derived from the preliminary laboratory phase of the trail, designed to evaluate the material’s safety and efficacy before its application in clinical settings.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.