Indiana University School of Dentistry Department of Endodontics / Introduction: Triple antibiotic paste (TAP, 1000 mg/ml) is composed of equal portions of ciprofloxacin, metronidazole and minocycline and is used as an intracanal dressing to disinfect the infected immature root canal during endodontic regeneration procedures. Lower concentrations of TAP have been recommended to minimize detrimental effects on pulp stem cells. TAP can be retained within the dentin matrix and its continual release confers an antibacterial effect to the dentin. Objective: The aim of this in vitro study was to investigate the residual antibacterial effect of dentin treated with various concentrations of TAP loaded into a gel system. Materials and Methods: Radicular dentin slabs were prepared from human teeth after obtaining IRB approval. The slabs were sterilized and treated with methylcellulose-based TAP of 100 mg/mL, 10 mg/mL, 1 mg/mL, 1.5% NaOCl, placebo paste with no TAP, or a positive control group with pure 1000 mg/mL TAP. Samples in each group were treated with the assigned TAP concentration for three weeks or immersed in 1.5% NaOCl for five minutes (n =18 per group). All samples were then irrigated with sterile water followed by 17% EDTA and incubated in phosphate buffered saline for either 2 or 4 weeks. Samples were then inoculated with Enterococcus faecalis and incubated for an additional 3 weeks. Biofilm formed on each sample was then dislodged and spiral plated to evaluate the bacterial colony-forming units. Data were analyzed using Fisher’s Exact tests and Wilcoxon rank sum tests (α = 0.05). Results: Dentin treated with 10, 100, or 1000 mg/mL of TAP demonstrated significant residual antibacterial effects up to four weeks. However, only 100 mg/mL TAP was able to completely prevent bacterial colonization after four weeks. No considerable residual antibacterial effect was observed in dentin treated with placebo gel, 1 mg/ml TAP or 1.5% NaOCl. Conclusion: At least 10 mg/mL of TAP loaded into a methylcellulose system is required to achieve a substantial residual antibacterial effect for four weeks.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic therapy in immature teeth with necrotic pulps triggers continued root development thereby improving the prognosis of these teeth. Several agents are under consideration for the disinfection and conditioning phases of this therapy. Triple antibiotic paste (TAP, i.e. equal parts of ciprofloxacin, metronidazole, minocycline) is used for canal disinfection and 17% EDTA solution is used for dentin conditioning. However, TAP and EDTA cause demineralization and their effect on surface loss and surface roughness of radicular dentin during regenerative procedures has not been quantified. Surface loss may be correlated with reduced tooth strength and surface roughness may be correlated with stem cell attachment. Objectives: The aim of this in vitro study was to quantitatively investigate the surface loss and surface roughness on human radicular dentin after treatment with two concentrations of TAP followed by EDTA. Materials and Methods: Human radicular dentin specimens were prepared from extracted human anterior teeth and randomized into six experimental groups. Group 1: saline control; Group 2: 17% EDTA; Group 3: TAP 1 mg/mL; Group 4: TAP 1 mg/mL and 17% EDTA; Group 5: TAP 1,000 mg/mL; Group 6: TAP 1,000 mg/mL and 17% EDTA for 5 minutes. After TAP is applied to Groups 3-6, all groups were incubated for 4 weeks. Then, groups 2, 4, and 6 were treated with EDTA for 5 minutes. Dentin surface loss (μm) and surface roughness (Ra, μm) were quantified after various treatments using non-contact and contact profilometry, respectively. Data were analyzed by one-way analysis of variance (α = 0.05) Hypothesis: It was hypothesized that there would be a significant difference in surface loss or surface roughness between at least two treatment groups. Results: All treatment groups showed significantly higher surface loss compared to untreated control. Dentin treated with 1g/mL TAP caused significant increase in surface loss and surface roughness compared to dentin treated with 1 mg/mL TAP. However, only 1g/mL TAP treated dentin showed significantly higher surface roughness compared to untreated control. The use of EDTA after both concentrations of TAP did not have significant additive effect on surface loss and surface roughness of dentin. Conclusion: The use of 1 mg/mL TAP can minimize surface loss and surface roughness of radicular dentin compared to higher concentrations. The use of EDTA after TAP may not cause additional surface loss and surface roughness of dentin.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic therapy in immature teeth with necrotic pulps triggers continued root development, thereby improving the prognosis of these teeth. Disinfection of the canal is accomplished with an intracanal medicament, such as triple antibiotic paste (TAP) composed of metronidazole, ciprofloxacin, and minocycline. A modified triple antibiotic paste (MTAP) that replaces minocycline with clindamycin has recently been suggested to avoid the tooth discoloration and potential demineralization from minocycline. The effect these pastes have on radicular dentin is unknown. Objectives: The aim of this study was to investigate the effects of two intracanal medicaments used during endodontic regeneration, TAP and MTAP, at concentrations of 1 g/mL and 1 mg/mL, on the microhardness and chemical structure of radicular dentin. Materials and Methods: Roots from extracted, unrestored, non-carious human premolar teeth were sectioned. An antibiotic paste (MTAP or TAP) or sterile water (control) was applied to treatment groups and stored for four weeks in 80-percent humidity at 37 °C. The effect of each paste on the microhardness of radicular dentin was measured using a Vickers Microhardness Tester (n = 17) to take three pretreatment and post-treatment measurements at both 500 µm and 1000 µm from the pulp-dentin interface. The chemical structure was assessed from dentin specimens treated with the same medicaments or sterile water for four weeks. After treatment, three measurements were taken on each specimen using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy to measure the phosphate/amide I ratios of dentin (n = 7). Results: The 1 g/mL of TAP or MTAP and the 1 mg/mL methylcellulose-based TAP caused significant reduction in microhardness of roots compared with untreated control roots at 500 µm and 1000 µm from the pulp-dentin interface. Furthermore, the methylcellulose-based 1 mg/mL TAP and MTAP caused significantly less reduction in microhardness compared with 1 g/mL TAP and MTAP. The 1 g/mL of TAP and DAP caused significantly lower phosphate/amide I ratios compared with other groups. Conclusion: The use of methylcellulose based 1 mg/mL of TAP and MTAP may minimize the reduction in microhardness of roots compared with the currently used 1 g/mL concentration of these antibiotics.

Indiana University-Purdue University Indianapolis (IUPUI) / Endodontic regeneration treatment of necrotic immature teeth has gained popularity in recent years. The approach suggests a biological alternative to induce a continuous root development. In this project, three in vitro experiments were conducted to investigate the effect of three medicaments used in endodontic regeneration on mechanical properties and chemical structure of radicular dentin. In the first experiment, we investigated longitudinally the effect of medicaments on the indentation properties of the root canal surface of immature teeth using a novel BioDent reference point indenter. A significant difference in the majority of indentation parameters between all groups was found after one-week and one-month application of medicaments (p<0.0001): triple antibiotic paste (TAP) > double antibiotic paste (DAP) > control > calcium hydroxide [Ca(OH)2]. The four-week exposure of dentin to TAP and DAP caused 43% and 31% increase in total indentation distance outcome, respectively. In the second experiment, we investigated longitudinally the effect of medicaments on the chemical structure of immature radicular dentin by measuring the phosphate/amide I ratios of dentin using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy. Phosphate/amide I ratios were significantly different between the four groups after one week, two weeks and four week application of medicaments (p<0.0001): Ca(OH)2-treated dentin > untreated dentin > DAP-treated dentin > TAP-treated dentin. In the third experiment, we investigated longitudinally the effect of medicaments on root fracture resistance and microhardness of radicular dentin. For the microhardness, the two-way interaction between group and time was significant (p<0.001). TAP and DAP caused a significant and continuous decrease in dentin microhardness after one and three month application, respectively. The three-month intracanal application of Ca(OH)2 significantly increased the microhardness of root dentin. The time factor had a significant effect on fracture resistance (p<0.001). All medicaments caused significant decrease in fracture resistance ranging between 19%-30% after three month application compared to one week application. The three medicaments used in endodontic regeneration caused significant change in the chemical integrity of the superficial radicular dentin and significantly affected the indentation properties of the root canal surface. Furthermore, the three month intracanal application of medicaments significantly reduced the fracture resistance of roots.

Indiana University-Purdue University Indianapolis (IUPUI) / Endodontic regeneration (ERP) has been successfully used in the treatment of traumatized immature teeth. The procedure has three essential steps: disinfecting the root canal (i.e. triple antibiotic paste (TAP) or double antibiotic paste (DAP)), provoking bleeding inside the canal to form a scaffold upon which pulp stem cells will be deposited and continue root growth, and creating a good coronal seal. Previous research has reported that antibiotic pastes (TAP and DAP) are cytotoxic to stem cells in the concentrations commonly used in endodontic regeneration (1000 mg/mL). To decrease the adverse effects on stem cells and increase the rate of success of the regeneration, defining appropriate antibiotic concentrations for ERP is critical. In this project, five in-vitro experiments were conducted to determine the breakpoint dilutions of both TAP and DAP medicaments, and to prepare a suitable novel pastes containing diluted TAP or DAP medicaments for ERP. In the first experiment, we compared the antibacterial effect of TAP, and DAP against early biofilm formation of Enterococcus faecalis (E. faecalis) and Porphyromonas gingivalis bacteria. In the second study, we investigated the antibacterial effect of various dilutions of TAP and DAP antibiotic medicaments against established E. faecalis biofilm. In the third experiment, we investigated longitudinally the residual antibacterial activity of human radicular dentin treated with 1000, 1 or 0.5 mg/ml of TAP and DAP. In the fourth study, we investigated the cytotoxic effect of various dilutions of TAP and DAP antibiotic medicaments on the survival of human dental pulp stem cells (DPSC). And in the fifth experiment, we investigated the antibacterial and cytotoxic effect of novel intracanal medicaments consisting of methylcellulose (MC) and/or propylene glycol (PG) mixed with 1mg/ml of TAP or DAP. 1 mg/ml of DAP or TAP medicaments had a significant antibacterial effect against early bacterial biofilm formation, and established bacterial biofilm. Furthermore, 1 mg/ml had a residual antibacterial activity comparable to 1000 mg/ml. The novel intracanal medicaments had comparable antibacterial effect to currently used medicaments (1000 mg/ml). Additionally, the novel intracanal medicaments significantly enhanced DPSC metabolic activity, compared to currently used medicaments in endodontic regeneration procedures.