Indiana University-Purdue University Indianapolis (IUPUI) / BACKGROUND: Dental erosion is a multifactorial condition that causes irreversible loss of dental hard tissues. Its development is highly influenced by saliva, with higher prevalence in hyposalivatory patients. There is no scientific consensus regarding the restorative treatment of choice for eroded teeth in such highly erosive conditions and to date, this has not been studied under in-vitro conditions. OBJECTIVES: To investigate the effect of erosion on direct tooth-colored restorations and adjacent enamel/dentin under different simulated salivary flow rates. METHODS: Bovine enamel and dentin specimens were prepared (n =16) and restored with the testing restorative materials, resin-composite (Filtek Z250), resin-modified glass ionomer (Fuji II LC), high viscosity glass ionomer cement (Fuji IX), and low viscosity glass ionomer cement (Fuji II).Then, submitted to an in-vitro erosion cycling model simulating different salivary flow rates (normal 0.5 ml/min and low 0.05 ml/min) and dental erosion protocols for 5 days. Surface loss of the restorative material and surrounding enamel/dentin surfaces were analyzed. A mixed-model ANOVAs and Sidak adjustment were used for statistical comparisons (p < 0.05). RESULTS: The surface loss was lower at 0.5 than at 0.05 ml/min, for all tested restorative materials except resin composite. Surface loss was higher in enamel and dentin adjacent to Filtek Z250 compared to Fuji II LC and Fuji IX, with no significant difference in enamel and dentin surface loss adjacent to Filtek Z250 and Fuji II. The restorations surface degradation was significantly lower for Filtek Z250 than for Fuji II, Fuji II LC, and Fuji IX, at both 0.5 and 0.05 ml/min; moreover, the surface loss was significantly lower for Fuji II LC than for Fuji II and Fuji IX, which did not differ from each other. CONCLUSION: Within the limitations of this study, it can be concluded that low salivary flow promoted higher erosive conditions. The use of Fuji II LC and Fuji IX may reduce erosive effects on enamel and dentin adjacent to restoration. Of the materials evaluated, resin-modified glass ionomer restoration may be the most suitable for restoration for patients at higher risk of erosion with low exposure to fluoride.

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) / 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)School of dentistry / BACKGROUND: Fluoride and stannous ions have been reported to be relevant for dental erosion prevention. However, their interaction with the acquired dental pellicle (ADP), a clinically relevant erosion protective factor, is not well known and needs to be investigated. OBJECTIVES: To investigate the anti-erosive properties of fluoride-containing solutions and stannous solutions on enamel and dentin surfaces with a previously formed ADP. To characterize the protein profile of the ADP treated with the test solutions. METHODS: Phase I tested four solutions: SnCl2/NaF, NaF, SnCl2 and deionized water (DIW) (as negative control). Forty bovine enamel and dentin specimens 104 (4x4x2 mm3) were prepared and randomly distributed into 4 groups (n = 10). The specimens were incubated in clarified human saliva (CHS) for 24 h for pellicle formation and then they were subjected to a cycling procedure that included a 5-min erosive challenge (0.3-percent citric acid, pH 2.6); a 2-min treatment with the solution (between 1st, 3rd and 6th cycles); a 2-h immersion in CHS, and overnight immersion in CHS. Cycles were repeated 6x/day for 5 days. The outcome measure was surface loss (SL) using profilometry. Phase II: Thirty-two (32) bovine enamel specimens (882 mm3) (n = 8) were similarly prepared and incubated in saliva for 24 h and then treated with the solutions for 2 min followed by CHS immersion for 2 h. This cycle was repeated 3x for one day. The pellicles formed and treated with the test rinse solutions were collected, digested, and analyzed for specific protein content using liquid chromatography electrospray ionization tandem mass spectrometry (LCESI-MS/MS). RESULTS: Phase I: for enamel, SnCl2/NaF, SnCl2, NaF solutions provided 89 percent, 67 percent, and 42 percent SL reduction respectively compared with the control, while in dentin they provided 60 percent, 23 percent, and 36 percent, respectively, all significant at p < 0.05. Phase II: Seventy-two (72) common proteins were identified in all groups, 30 exclusive to DIW, 20 to SnCl2/NaF, 19 to NaF, and 13 to SnCl2. SnCl2/NaF increased the abundance of pellicle proteins than each one alone. CONCLUSION: SnCl2/NaF showed the best anti-erosive effect on both enamel and dentin. The findings suggest that the composition of acquired pellicle changes with different solutions, which may be related to their anti-erosive effect.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic therapy in immature teeth promotes continuation of root development and likely increases the prognosis of these teeth. The use of double antibiotic paste (DAP), equal parts of ciprofloxacin and metronidazole, followed by the dentin conditioner, ethylenediaminetetraacetic acid (EDTA), has been suggested for canal disinfection and facilitation of stem cell attachment/proliferation, respectively. However, the effect is unknown when all these agents are used on on radicular dentin surfaces to facilitate the level of stem cell proliferation. Objectives: The aim of this in-vitro study is to compare the proliferation of human dental pulp stem cells (hDPSCs) on human radicular dentin treated with two different concentrations of DAP followed by EDTA. Materials and Methods: Human premolars and incisors were prepared into standardized polished 4 mm x4 mm radicular dentin specimens. Groups of specimens were treated with DAP 500 mg/mL, DAP 1 mg/mL, DAP 500 mg/mL followed by 17-percent EDTA, DAP 1 mg/mL followed by 17-percent EDTA; 17% EDTA, or no treatment. All groups treated with antibiotics were incubated with DAP at 37°C for one week. All specimens were washed with distilled water. The hDPSCs were seeded across all specimens and unattached cells were collected after 24 hours. LDH assay was completed on unattached cells for quantification. Three days after attachment, WST viability and LDH cytotoxicity assays were performed. Hypothesis: There is no significant difference in hDPSC viability, unattachment, and cytotoxicity on dentin specimens treated with DAP and 17-percent EDTA. Clinical Significance: These results can be used to help identify the best treatment concentrations when using DAP and/or EDTA to promote endodontic regeneration. Results: The results demonstrated significantly less viability of hDPSCs on specimens treated with 500 mg/mL DAP with and without 17-percent EDTA. Groups treated with 1 mg/mL DAP, 1 mg/mL DAP and 17-percent EDTA, and 17-percent EDTA alone had no statistically significant difference in viability compared with control untreated dentin. The results of the unattached cells from the LDH demonstrated that cells from the specimens treated with solely 500 mg/mL and 1 mg/mL DAP had significantly higher levels of unattached cells when compared with all other groups. The LDH assays in summation with the WST assays showed a trend of a lack of proliferation on groups treated with 500 mg/mL DAP with and without 17-percent EDTA. Conclusions: Paste-like concentrations (500 mg/mL) of DAP are detrimental to hDPSC viability, whereas the present study supports the use of low-concentration antibiotics consistent with current recommendations for intracanal medicaments used during endodontic regenerative procedures.

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 (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.