Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontics (RE) is a treatment alternative for the infected immature tooth to establish an environment in the canal that enables continued root development and the growth of pulp or pulp-like tissue within the canal. A scaffold created in the canal encourages the formation of vital tissue. The porcine sub-intestinal-submucosa (SIS) membrane, Dynamatrix®, has the potential to serve as an endodontic scaffold. Research at Indiana University School of Dentistry (IUSD) has shown that Dynamatrix® can support the growth of human dental pulp stem cells (HDPSC) and human pulp fibroblasts (HPF). Positive angiogenic cytokine profiles were seen after these cells were seeded on Dynamatrix®. Endothelial cells play an important role in the formation of blood vessels and are a source of angiogenic cytokines. Exposure of these cells to DynaMatrix® may result in a positive angiogenic profile for both cytokines and matrix metalloproteinases (MMPs). Objective: The aim of this in-vitro study was to investigate if the exposure of human endothelial cells to the DynaMatrix® membrane would result in differences in the expression of cytokines and MMPs that play roles in angiogenesis. Materials and Methods: Human endothelial cells (HUVECs) were obtained from American Type Culture Collection (ATTC, Manassas, VA) and used in this study. Groups were established as follows: (a) Group 1: HUVECs seeded in culture media only, (b) Group 2: DynaMatrix® membrane incubated alone in the serum-media without any cells, and (c) Group 3: HUVECs seeded on DynaMatrix® membranes. After 72 hours of incubation, the conditioned media were collected and analyzed for the expression of 20 angiogenic cytokines and MMPs utilizing cytokine and MMP protein arrays. The density of each cytokine and MMP expressed was measured, averaged, and statistically analyzed by ANOVA. Results: Exposure of human umbilical vein endothelial cells (HUVECs) to the DynaMatrix® membrane resulted in a positive angiogenic profile for both cytokines and MMPs. Conclusion: This work furthers the evidence for the potential of DynaMatrix® to serve as a more predictable scaffold in RE.

Indiana University-Purdue University Indianapolis (IUPUI); Graduate Endodontics, Masters degree thesis / We evaluated the antibacterial stability (shelf life) of a new radiopaque double antibiotic paste (RoDAP) loaded in a methylcellulose system with 30% w/v barium sulfate against biofilm collected from an immature tooth with necrotic pulp. Uniform radicular dentin specimens were infected with bacterial biofilm obtained from an immature tooth with a necrotic pulp and incubated anaerobically for three weeks. These samples were randomly divided into 6 experimental groups (n=7) and treated for 1 week at three time points of aged radiopaque DAP: 0 months, 3 months, and 6 months. Group 1: 1mg/mL RoDAP treatment. Group 2: 10 mg/mL RoDAP treatment. Group 3: Calcium hydroxide (Ca(OH)2) treatment. Group 4: Methylcellulose with barium sulfate. Group 5: No treatment. Group 6: No bacteria or treatment. The samples were rinsed with sterile saline to detach biofilms and then spiral plated using a biofilm disruption assay. Statistical analyses were performed using Wilcoxon rank-sum tests and Wilcoxon signed rank tests with fixed effects for treatment, time, and the treatment-by-time interaction. Treatment of infected dentin with 1 mg/mL RoDAP, 10 mg/mL RoDAP, and Ca(OH)2 demonstrated significant and substantial antibiofilm effects in comparison to untreated control groups or groups treated with placebo paste after 0, 3, and 6 months of aging. Calcium hydroxide, however, showed slightly less antibiofilm activity after 6 months of aging when compared to 0 months and 3 months of aging. This difference was statistically significant (p > 0.05). In conclusion, both concentrations of RoDAP maintained full antibacterial efficacy after 6 months of aging, while calcium hydroxide lost some antibacterial activity after a shelf life of 6 months.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures require adequate disinfection of immature teeth with necrotic pulps. Regeneration endodontic procedures have been shown to increase width and length of dentin after disinfection of the root canal system with various antibiotic pastes such as double antibiotic paste (DAP). DAP is composed of an equal ratio of metronidazole and ciprofloxacin. After the root canal system is disinfected with DAP, it is later flushed out with 17-percent EDTA. There is a need for a radiopaque component in the DAP to facilitate proper placement. Following disinfection with DAP, a residual antibacterial effect is formed in dentin. It is not known if this effect is modified by making DAP radiopaque. Objectives: The residual antibacterial effects of a radiopaque double antibiotic paste against a bacterial isolate obtained from an immature tooth with necrotic pulp on radicular dentin will be explored utilizing various strengths of DAP (1 mg/mL, 10 mg/mL, and 25 mg/mL) after 1 week and 4 weeks. Materials and Methods: Dentin samples of 4x4-mm will be prepared from previously extracted human posterior teeth. The samples will be assigned to seven treatment groups in a random fashion (G1 will be treated with 25-, G2-10, and G3-1 mg/mL of radiopaque DAP. G4 will be treated with the radiopaque filler without DAP. G5 will be treated with Ca(OH)2 and G6 sterile water with bacterial biofilm, and G7 will be sterile water). The groups will be treated for both 1 week 4 weeks. These samples will then be stored in a phosphate buffered saline (PBS) solution for 1 week and then inoculated with the cultured bacterial isolate from an immature tooth with necrotic pulp. The specimens will be incubated for three weeks to permit adequate formation of a biofilm. The biofilm will be detached, diluted and spirally plated onto blood agar plates and incubated for a total of 48 hours in 5-percent CO2 at 37°C. The number of CFUs/mL will be counted using an automated colony counter. A two way ANOVA and Fisher’s Protected Least Significant Differences test using 5-percent significance level will be used to evaluate the resulting data. Expected outcome: A residual antibacterial effect will be observed on radicular dentin samples when treated with radiopaque DAP. Conclusion: Using a radiopaque DAP that provides residual antibacterial effects, further awareness into regenerative endodontic procedures will be gained leading to alterations in treating such cases. / 2020-06-30

Indiana University-Purdue University Indianapolis (IUPUI) / EFFECTS OF DYNAMATRIX® ON ANGIOGENIC CYTOKINE EXPRESSION FROM HUMAN DENTAL PULP FIBROBLASTS: AN IN VITRO STUDY by Joseph Benjamin Adams Indiana University School of Dentistry Indianapolis, IN Introduction: An exogenous scaffold may lead to more predictable pulp tissue regeneration and continued root formation in a regenerative endodontic procedure. DynaMatrix® is a natural membrane scaffold made of porcine small intestine, currently used in periodontal regenerative surgeries. Objective: The purpose of this study was to investigate if human dental pulp fibroblasts (HDPFs) seeded on DynaMatrix® membrane would result in an increase in the expression of angiogenic cytokines. Materials and Methods: HDPFs (75,000 per well) were seeded in 6-well plates. Three groups were tested: Group 1 (C): HDPFs in 70 media only; Group 2 (M): DynaMatrix® (Cook Biotech, Indianapolis, IN) alone in media; and Group 3 (C+M): HDPFs seeded on DynaMatrix® membranes. After 72 hours of incubation in serum positive, the conditioned media were collected and analyzed for the expression of 20 angiogenic cytokines utilizing RayBiotech Inc., arrays per the manufacturer’s instruction. The data were analyzed by ANOVA. Results: Group M was significantly higher than C for bFGF (p = 0.0023). C+M was significantly higher than M for ANG (p = 0.0104); GRO (p = 0.0003); IFN-γ (p = 0.0023); IL-6 (p = 0.0003); IL-8 (p = 0.0003); Leptin (p = 0.0003); MCP-1 (p = 0.0104); TIMP-1 (p = 0.0190); TIMP-2 (0.0123). C was significantly higher than C+M for ANG (p = 0.0104); MCP-1 (p = 0.0104); and THPO (p = 0.0308). Cytokines such as b-FGF, ANG, and leptin promote angiogenesis, and stimulate migration and proliferation of cells. Conclusion: The cytokine expression profile from the cells seeded on DynaMatrix® suggests that it might be a suitable scaffold for regenerative endodontic procedures. It could improve vascularization by increasing angiogenic cytokines in the microenvironment of the treated root canal and supporting tissue regeneration.

Indiana University School of Dentistry / Introduction: The goal of nonsurgical root canal therapy is to reduce the bacterial load within an infected root canal system, and the subsequent objective is to prevent or treat apical periodontitis. Clinical studies have shown more expedient healing of apical periodontitis treated with electromagnetic wave stimulation (EMS) as compared to apical periodontitis not treated with EMS. Stimulation of osteoblasts and growth factors has been shown when EMS was applied to rat calvaria, resulting in increased bone healing. Objective: The purpose of this vitro study was to evaluate the effects of EMS on the proliferation and differentiation of osteoblasts. Using primary neonatal calvaria osteoblast-lineage cells, the effects of different EMS regimens on proliferation, alkaline phosphatase (ALP) activity, and mineral deposition were determined. Materials and Methods: EMS regimen included currents of 0mA, 0.1mA, 1mA, and 10mA delivered for five consecutive 1s pulses per day for one, two, and three days. Cell proliferation was assayed after 1 or 2 days using an MTS assay. Alkaline phosphatase activity and mineral deposition were assayed after culturing the cells in osteogenic media containing ascorbic acid and -glycerol phosphate for 7 days. Comparisons were performed using analysis of variance, with a 5% significance level. Results: There was no statistically significant differences noted in MTS proliferation and mineral deposition between the experiment EMS treatment groups of 0.1, 1.0, and 10.0 mA compared to the control group of 0 mA current on calvaria-derived osteoblast. While there were no statistically significant differences noted in ALP activity in the 0.1, and 1.0 mA EMS groups, compared to 0 mA control, alkaline phosphatase activity was significantly increased in the 10 mA EMS group. Conclusion: There was no significant differences in MTS proliferation and mineral deposition of the EMS group compared to the control group. However, 10 mA EMS favored increased ALP activity suggesting EMS can promote matrix maturation by osteoblasts. Additional in vitro experimental studies, including different stem cell populations, culture duration and EMS treatment regimens are needed to understand the mechanism of action of EMS for future applications in regenerative endodontics.

Indiana University-Purdue University Indianapolis (IUPUI) / The effects of hydroxyapatite in a DAP-loaded MC carrier on dental pulp stem cells Introduction: Regenerative endodontic procedures (REP) require disinfection techniques to eliminate bacteria from the infected immature root canal system and promote new growth of the pulp-dentin complex. Double antibiotic paste (DAP), a mixture of ciprofloxacin and metronidazole, has shown efficacy in doing so while minimizing cytotoxicity on dental pulp stem cells (DPSC). Stem cells, scaffolding, and growth factors are necessary in the maturation, proliferation, and differentiation of mesenchymal stem cells into the root canal system. Nano-hydroxyapatite (n-HA) has a history of biocompatibility and, in addition, has shown promising effects as a tissue bioengineering material. Objective: The aim of this in vitro study was to investigate the proliferation and mineralization of DPSC in the presence of 1% DAP and methylcellulose (MC) with varying concentrations of nano-hydroxyapatite. Materials and Methods: DPSC were plated in 24-well plates containing culture media. The next day, semi-permeable 0.1 mm Transwell chambers were inserted into the wells to separate the reservoirs for medicaments. Treatment paste composed of methylcellulose containing 1% DAP with either 0.25%, 0.50%, or 1.0% nano-hydroxyapatite was added along with culture media. Methylcellulose alone and calcium hydroxide (Ultracal) were used as control groups. After 3 days, cells were evaluated for cytotoxic effects using an MTS proliferation assay (n = 10, in triplicate). DPSCs were also cultured with these medicaments for 7 days in osteogenic media and evaluated for alkaline phosphatase (ALP) activity and mineralization activity (n = 13, in triplicate). Comparisons between groups for differences in mineralization, BSA, and ALP activity were performed using analysis of variance (ANOVA), with different variances allowed for each group and a random effect included in the model to account for correlation within each of the three trials. A simulation-based model was used to adjust for multiple comparisons. Results: Addition of n-HA treatment groups increased mineralization significantly greater than calcium hydroxide, with MC alone and MC+DAP+0.5% HA providing the greatest effect. Regarding ALP, all HA concentrations performed significantly greater than MC and DAP concentrations. Proliferation demonstrated similar metabolic activity in all experimental groups with few comparisons significant. Conclusion: The challenge in REPs is to maintain survival, and preferably promote the proliferation and development of DPSCs into the pulp-dentin complex with a consistent treatment outcome. The combination of DAP with hydroxyapatite may allow for both disinfection and improved mineralization and cellular differentiation. This contribution has shown significant ability to increase stem cell differentiation into an osteogenic lineage as well as calcium deposition, indicating end goal results of regenerative procedures.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures (REPs) are a type of endodontic treatment aimed at replacing damaged tooth structures, including dentin and root structures, as well as cells of the pulp-dentin complex. Double antibiotic paste (DAP) has been shown to be efficacious in achieving disinfection of the root canal system while minimizing cytotoxicity to dental pulp stem cells (DPSCs). Hydroxyapatite (HA) is an extracellular, mineralized component of bone that has shown much promise as a scaffold in the field of regenerative medicine. Objective: The objective of this study was to evaluate the effects of HA in a DAP loaded methylcellulose (MC) carrier on the differentiation and mineral deposition of DPSC over time. Materials and Methods: DPSCs were plated in 24-well plates with culture media. The following day, semi-permeable 0.1 m chambers were inserted into the wells to separate the reservoirs and permit delivery of medicaments. 100 L treatment paste composed of MC with 1% DAP and either 0.5% or 1.0% nano-HA was added, followed by additional culture media. After 3 days of treatment, medicaments were removed and DPSCs were cultured for an additional 9 days with replacement of media every 3-4 days. At Day 12, DPSCs were evaluated for alkaline phosphatase (ALP) activity using a biochemical assay and mineral deposition using an Alizarin Red S Ca2+ staining assay (4 wells/group). Comparisons between groups were performed using one-way analysis of variance (ANOVA) with a 5% significance level used for all tests. Results: A trend towards increased ALP and mineral deposition activity was noted among the groups with HA added to DAP with MC. Although these trends were not statistically significant, a trend towards increased ALP and mineral deposition was observed after 3-day medicament exposure. The results were similar to previous findings using 7-day medicament treatments. Conclusion: The addition of HA showed a trend towards improved differentiation and mineral deposition of DPSCs compared to DAP with MC. Although additional studies are required, these results showed suggest that even with a shortened treatment time, increased differentiation and mineral deposition of DPSCs may be possible. This study provides additional support that low concentration DAP in a MC carrier has potential application in regenerative endodontic procedures. The novel addition of HA may provide additional osteogenic potential.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures are an effective treatment option for immature teeth with infected necrotic pulps to allow for healing and potential continued root development, yet challenges to ideal treatment outcomes remain. Consistent development of root length and width of dentin remains a challenge, as does development of the pulp-dentin complex. Previous in vitro studies have assessed the role of different growth factors and bioactive molecules in combination with scaffolds to potentially facilitate continued development of the pulp-dentin complex using dental pulp stem cells (DPSCs). The proline-rich tyrosine kinase 2 (Pyk2) is linked with osteoblast activity and the regulation of bone mass. Further, the Pyk2 inhibitor PF-4618433 (PF-46) has been shown in previous studies to enhance osteoblast activity and mineral deposition in vitro. However, whether Pyk2 targeting promotes the osteogenic differentiation of DPSCs remains unknown. Objective: The purpose of this study was to investigate the effect of a Pyk2 inhibitor, PF-46, on the proliferation, differentiation, and mineralization of human DPSCs. Materials and Methods: Human DPSCs were cultured in 24-well plates with α-MEM with 10% FBS, and containing 0 μM (vehicle control) or 0.1 μM, 0.3 μM, or 0.6 μM PF-46. Fresh media and treatments were replaced every 2-3 days. After 1 day incubation, cytotoxic effects were evaluated by using an MTS proliferation assay. After 4 days of treatment, direct cell counting was performed. To induce osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media and the DPSCs were cultured with PF-46 for 14 days. Then, an alkaline phosphatase (ALP) assay and mineral deposition assay were performed. Differences between treatment groups were analyzed by a one-way ANOVA followed by pair-wise tests conducted using Tukey’s multiple comparisons procedure with a 5% significance level. Results: The 0.6 μM PF-46 group had a significantly higher cell count, ALP activity and mineral deposition when compared to 0 μM PF-46. The 0.1 and 0.3 μM PF-46 groups also had significantly higher ALP activity compared to the 0 μM PF-46 group after 14 days of incubation. There was a general trend of increased differentiation and mineral deposition as the concentration of PF-46 increased from 0.1 μM to 0.6 μM. Conclusion: There was a general concentration-dependent increase in cell count, differentiation, and mineral deposition by human DPSCs as the concentration of PF-46 increased from 0 μM up to 0.6 μM, with the highest activity observed with 0.6 μM PF-46. Although further research is needed, these results suggest that strategies that target Pyk2 may potentially be used to improve the osteogenic differentiation of DPSCs to aid endodontic regeneration.

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.