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-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) / 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.