Regenerative Capacity of Human Dental Pulp and Apical Papilla Cells after Treatment with a 3-Antibiotic Mixture

doi:10.1016/j.joen.2013.09.027

Journal of Endodontics

Volume 40, Issue 3, March 2014, Pages 399-405

https://doi.org/10.1016/j.joen.2013.09.027 Get rights and content

Abstract

Introduction

A 3-antibiotic combination (3Mix) has been widely used in regenerative endodontics. Recent studies recommend that a safe concentration of 3Mix is in the range of 0.39 μg/mL and 1 mg/mL because higher concentrations may limit tissue regeneration. The aim of this study was to determine the regenerative capacity of isolated human dental pulp cells (DPCs) and apical papilla cells (APCs) after a 7-day treatment with selected doses of 3Mix.

Methods

Primary human DPCs/APCs from the third passage were divided into control and experimental groups. In the control group, cells were cultured in regular complete media. In the experimental group, cells were cultured in complete media containing 0.39 μg/mL or 1 mg/mL of 3Mix for 7 days. After the treatment period, the media were changed, and the cells were further tested for proliferation and differentiation potential. For cell proliferation, a colorimetric qualification of 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide was used on days 1, 3, 5, and 7. For differentiation potential, a dentinogenic differentiation medium was added into treated cells and cultured for 7, 14, and 21 days. Results were analyzed using quantitative alizarin red S staining and real-time reverse-transcription polymerase chain reaction.

Results

After 7 days of treatment, 100% cell death was discovered in the 1-mg/mL 3Mix group. The proliferative capacity of 0.39 μg/mL 3Mix-treated DPCs and APCs was significantly lower than that of untreated cells at all time points (P < .05). Mineralized nodule formation was found both in the 3Mix-treated and control groups, but it was significantly less in the 3Mix-treated groups at 7, 14, and 21 days (P < .01). Quantitative reverse-transcription polymerase chain reaction showed no statistically significant difference (95% confidence interval) in bone sialoprotein, alkaline phosphatase, and dentin matrix protein 1 gene expression in either 3Mix-treated DPCs or APCs compared with control groups.

Conclusions

One milligram per milliliter of 3Mix had strong toxicity to DPCs/APCs when applied for 7 days, whereas 0.39 μg/mL 3Mix showed no toxicity but still affected cell proliferation and mineralization potential. However, no differences in dentinogenic gene expressions were observed between the 3Mix-treated and untreated groups.

Section snippets

Patient Recruitment

This study was approved by the Human Experimentation Committee of the Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand. After verbal and written informed consent, nonpathologic impacted third molars with/without immature roots from healthy patients (aged 18–25 years) were extracted. The teeth were rinsed using sterile normal saline solution and stored on ice in separate containers with serum-free media.

Culture of Primary Human DPCs and APCs

To obtain pulp tissue, teeth were soaked and inverted in 5.25% sodium

Proliferative Capacity of 3Mix-treated and Untreated DPCs/APCs

No DPCs/APCs from experimental group A (1 mg/mL 3Mix treatment for 7 days before assessment) survived after the treatment period. Cell morphology had changed on the fourth day of 3Mix treatment, and the cells dissolved afterward.

There were significant differences (P < .05) in the proliferative capacity of DPCs at all time points in group B (0.39 μg/mL 3Mix treatment for 7 days before assessment) compared with the control group. To clarify the ratio of cell proliferation capacity, the

Discussion

The consequences of DPCs and APCs after exposure to 3Mix at 1 mg/mL and 0.39 μg/mL for 7 days were evaluated. The total cell death occurred when 1 mg/mL of 3Mix, an LC50 dose (14), was used to culture both cell types for 7 days. The proliferation assay revealed that 0.39 μg/mL of 3Mix-treated cells had a significantly lower proliferation rate than untreated cells. Mineralized matrix formation was observed in both groups, but it was lower in the 3Mix-treated groups than in the control groups.

Conclusions

After treatment with 3Mix, even at a very low concentration, it still had effects on the proliferative capacity and mineralized matrix formation of DPCs and APCs in vitro. Alternative regimens for regenerative endodontics, including other influencing factors, still need to be clarified to promote appropriate pathways for dental tissue regeneration.

Acknowledgments

The authors deny any conflicts of interest related to this study.

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The viability of human dental pulp cells and apical papilla cells after treatment with conventional calcium hydroxide and nanoparticulate calcium hydroxide at various concentrations
2023, Saudi Dental Journal
Nanoparticulate Ca(OH)₂ had greater antibacterial effect than conventional Ca(OH)₂. Conversely, a study reported that nanoparticulate Ca(OH)₂ had toxicity against murine fibroblast. However, the study of nanoparticulate Ca(OH)₂, involving human dental pulp cells (DPCs) and apical papilla cells (APCs) is lacking. The aim of this study is to compare the effects of conventional Ca(OH)₂ and nanoparticulate Ca(OH)₂ on the viability of DPCs and APCs.
Primary human DPCs/APCs from the 3rd to 5th passage were divided into control and experimental groups. In the control group, cells were cultured in complete media. In the experimental group, cells were cultured in complete media containing 10, 100, or 1000 μg/mL of either conventional Ca(OH)₂ or nanoparticulate Ca(OH)₂ for 1, 3, 5, and 7 days. After the treatment period, the cells were tested for viability using MTT assay.
DPCs treated with conventional Ca(OH)₂ in all concentrations at day 5 revealed significantly higher proliferation compared to nanoparticulate Ca(OH)₂ treated groups. In additions, DPCs treated with 1000 µg/ml nanoparticulate Ca(OH)₂ at day7 were significantly lower proliferation compared to DPCs treated with conventional Ca(OH)₂. In contrast, APCs treated with 1000 µg/ml nanoparticulated Ca(OH)₂ were significantly higher proliferation than APCs treated with 1000 µg/ml conventional Ca(OH)₂ at day7.
Nanoparticulate Ca(OH)₂ increased the viability of APCs and can be an alternative choice of intracanal medication for regenerative endodontic procedures. However, Nanoparticulate Ca(OH)₂ exerted some effects on DPCs. The use of nanoparticulate Ca(OH)₂ has no advantages over the conventional Ca(OH)₂ for vital pulp therapy.
A prospective case series in regenerative endodontics: The effective use of diluted antibiotic hydrogels in endodontic regeneration procedures
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to investigate the effectiveness of diluted antibiotic hydrogels in endodontic regeneration procedures.
One conventional treatment (calcium hydroxide) and two experimental treatments containing 1 mg/mL antibiotic in gel (MC-TAP and MC-DAP) were prepared for this study. The effect of these medicaments on the proliferation and differentiation potential of dental pulp stem cells (DPSCs) was examined before their clinical use, using WST and ALP assays. Twenty-two teeth diagnosed with necrotic immature roots were treated with calcium hydroxide, MC-TAP or MC-DAP using the endodontic regeneration protocol as described by the American Association of Endodontists (AAE). Teeth were examined after application of the medicament, at the time of regeneration and after 12–24 months.
All treated teeth showed resolution of signs and symptoms shortly after treatment with the different medicaments. However, signs and symptoms returned a few months after initial treatment in clinical cases treated with MC-DAP. All successfully treated teeth examined after 12–24 months were free of signs and symptoms, but vitality was not achieved.
Low concentration antibiotic gel containing triple antibiotic paste (MC-TAP) can be successfully used for endodontic regeneration procedures.
Antimicrobial Activity and Biocompatibility of Antibiotic-Loaded Chitosan Hydrogels as a Potential Scaffold in Regenerative Endodontic Treatment
2020, Journal of Endodontics
The objective of this study was to determine the effectiveness of several antibiotic-loaded hydrogel scaffolds against Enterococcus faecalis, as well as their ability to stimulate proliferation and mineralization of dental pulp stem cells.
Fibrin (Fg) or chitosan-fibrin hydrogels (Ch) were prepared using 12.5 mg/mL fibrinogen and 0.4% (w/v) chitosan. Triple antibiotics, clindamycin-modified triple antibiotic paste, or double antibiotics were loaded in gels (1 mg/mL). Antibacterial effect against E. faecalis biofilm was determined by using colony-forming units (CFUs) and confocal laser scanning microscope (CLSM). Cell viability and morphology were determined by loading cells into different gels at 7 and 14 days using the water-soluble tetrazolium salt-1 cell viability assay and Live & Dead cell analysis. Mineralization was detected by using alkaline phosphatase and alizarin red staining activity.
Antibiotic-loaded Fg gel and Ch gel alone without antibiotics resulted in a significant reduction in CFUs compared with the positive control (P < .05). When antibiotics were loaded in Ch gel, there were no CFUs detected in any groups (P < .05). CLSM images showed dense red areas with mostly dead bacteria on the dentin surface in antibiotic-loaded Ch groups, which showed significantly less live bacteria compared with the other groups (P < .05). Triple antibiotic-loaded Fg and Ch gels resulted in a dramatic decrease in the mineralized nodule formation compared with all other gel groups (P < .05). Ch hydrogels resulted in round cell morphology up to 7 days. Ch alone or with double antibiotic paste showed more cell spreading with spindle-shaped morphology at 14 days and higher alkaline phosphatase activity compared with other antibiotic-loaded Ch groups (P > .05).
Double antibiotic-loaded Ch gel appears to enhance the antibacterial properties while maintaining higher cell viability, cell spreading, and mineralization activity, compared with all the other scaffolds investigated.
Effects of radiopaque double antibiotic pastes on the proliferation, alkaline phosphatase activity and mineral deposition of dental pulp stem cells
2020, Archives of Oral Biology
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Moreover, the MC-BaSO4 DAP1 was not significantly different to all the control groups. Numerous studies explored the biocompatibility of stem cells against various intracanal antibiotic medicaments and suggested the use of low concentrations of antibiotics to minimize their cytotoxic effects (Althumairy, Teixeira, & Diogenes, 2014; Chuensombat et al., 2013; Phumpatrakom & Srisuwan, 2014; Ruparel et al., 2012). However, these published studies explored pure antibiotic pastes mixed with water at higher concentrations, or used diluted antibiotic solutions.
The aim of this study was to investigate the effects of two radiopaque agents, barium sulfate (BaSO₄) or zirconium oxide (ZrO₂) in double antibiotic paste (DAP), on the proliferation and mineral deposition of human dental pulp stem cells (DPSC).
Radiopaque antimicrobial medicaments composed of methylcellulose (MC) thickening polymer with BaSO₄ or ZrO₂ and either 1 or 5 mg/mL DAP (equal portions of metronidazole and ciprofloxacin) were used to investigate DPSC proliferation after 3 days, and alkaline phosphatase (ALP) activity and mineral deposition after 7 and 14 days. Radiopaque agents without DAP and Ca(OH)₂ were used as controls.
MC-BaSO₄ DAP and MC-ZrO₂ DAP at 1 or 5 mg/mL had no adverse effect on DPSC proliferation, compared to the media and MC controls. MC-ZrO₂ (DAP-free) greatly increased ALP activity after 7 days. DPSC mineral deposition was modestly reduced at 7 days by MC-BaSO₄ DAP and MC-ZrO₂ DAP, but not by DAP-free radiopaque agents, and was most reduced by 5 mg/mL DAP in the 14-day cultures.
MC-BaSO₄ or MC-ZrO₂ medicaments containing up to 5 mg/mL of DAP supported the proliferation and early osteogenic differentiation of DPSC. Low DAP concentrations and short culture times led to more favorable effects on ALP activity and mineral deposition by DPSC. The findings suggest that radiopaque agents added for the purpose of detecting whether medicaments occupy the full extent of the root canal may have clinical applications. Radiopaque antibiotic medicaments containing low DAP concentrations may be an alternative to Ca(OH)₂ for regenerative endodontic procedures.
A review of tooth discoloration after regenerative endodontic therapy
2016, Journal of Endodontics
Citation Excerpt :
The AAE guidelines (20) recommend either calcium hydroxide or a low concentration of TAP as an intracanal medicament. A recent study suggested a safe concentration in the range of 0.39 μm/mL and 1 mg/mL because higher concentrations may limit tissue regeneration (100). In a more recent in vitro study published after the AAE guidelines were revised, DAP showed a significantly longer residual antibacterial effect compared with the same concentrations of TAP in human radicular dentin specimens (101).
The American Association of Endodontists clinical considerations for regenerative therapy (April 2015) advise of the risk of the possible adverse effect of staining of teeth. It is widely acknowledged that before these guidelines were developed there was no standardized protocol for regenerative therapy, and different approaches using different materials have been reported. The purpose of this review was to undertake a systematic review of published cases to determine the incidence of reported discoloration and examine whether there was any particular association with any material.
A PubMed search was performed using key words for regenerative endodontic therapy consistent with prior published systematic reviews.
There were 80 studies identified with 379 teeth treated. Many of the studies did not report on the presence or absence of discoloration. It was noted that there was a strong association of discoloration with the use of triple antibiotic paste containing minocycline; however, discoloration was also noted when other materials were used. Unfortunately, bleaching of stained teeth was not predictably achieved in some studies.
The use of triple antibiotic paste with minocycline as the intracanal medicament should be reconsidered. Either calcium hydroxide or the double antibiotic paste of metronidazole and ciprofloxacin may limit discoloration. Mineral trioxide aggregate was also associated with discoloration. However, there is little evidence of an alternative superior material as a coronal barrier. Because discoloration is a patient-orientated outcome, further research should document and identify the incidence of discoloration in regenerative endodontic treatment.
Attachment Ability of Human Apical Papilla Cells to Root Dentin Surfaces Treated with Either 3Mix or Calcium Hydroxide
2016, Journal of Endodontics
Citation Excerpt :
Moreover, from our results, it can be speculated that the effect of APC attachment might be concentration-dependent, because higher cell numbers were noticed when a lower concentration of 3Mix was used. Similar results have been reported, suggesting that high concentrations of 3Mix create more destructive effects (4–6). From our SEM study, 3Mix-treated dentin had fewer cells on the surface.
Tissue regeneration and root continuation are the ultimate goals in regenerative endodontics. Apical papilla cells (APCs) have been hypothesized to play roles in those processes. Therefore, preservation of cell vitality and promoting initial cell attachment seem to be crucial steps. The purposes of this study were to investigate the attachment ability and morphology of viable human APCs by using fibronectin immunofluoresence, alamarBlue assay, and scanning electron microscopy, when APCs were grown on human root dentin surfaces treated with 3Mix or calcium hydroxide (Ca(OH)₂) at different concentrations.
Human root dentin slices were divided into 6 groups: (1) control, (2) 3Mix 0.39 μg/mL, (3) 3Mix 100 μg/mL, (4) 3Mix paste, (5) Ca(OH)₂ 1 mg/mL, and (6) Ca(OH)₂ 1000 mg/mL. All samples were separately treated for 7 days and final rinsed with 17% EDTA. Then primary human APCs were seeded. Fibronectin immunofluorescence was used to evaluate the attachment ability of APCs on treated dentin. A vitality assay by using alamarBlue was monitored at specific time intervals. The morphology of the cells on the dentin was observed under scanning electron microscopy.
The lowest number of fibronectin-positive cells was observed on root dentin treated with 3Mix paste (P < .05). The 3Mix at 0.39 and 100 μg/mL did not affect the amount of APC attachment, whereas the viability of APCs on the dentin surface was significantly lower in the 100-μg/mL 3Mix-treated group than in the negative control group (P < .05). Both concentrations of Ca(OH)₂ induced APC attachment (P < .05). Moreover, only cells grown on the surfaces treated with Ca(OH)₂ exhibited cytoplasmic processes.
Human root dentin treated with 3Mix paste had significantly lower APC attachment. The 3Mix at 0.39 μg/mL and 100 μg/mL had no significant negative effect on APC attachment on dentin. Higher numbers of cells attaching on dentin were observed in calcium hydroxide–treated groups.

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Basic ResearchRegenerative Capacity of Human Dental Pulp and Apical Papilla Cells after Treatment with a 3-Antibiotic Mixture

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

Patient Recruitment

Culture of Primary Human DPCs and APCs

Proliferative Capacity of 3Mix-treated and Untreated DPCs/APCs

Discussion

Conclusions

Acknowledgments

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Basic Research
Regenerative Capacity of Human Dental Pulp and Apical Papilla Cells after Treatment with a 3-Antibiotic Mixture