Introduction: Millions of people suffer from dental trauma and/or caries every year. In these situations, pulp tissue gradually becomes inflamed and, if not treated, tissue death will eventually cause pain and/or a localized dental infection [1]. Root canal irrigation with sodium hypochlorite associated with antibiotic mixtures (e.g., triple antibiotic paste, TAP) has demonstrated to eliminate bacterial infection [2]. Regrettably, antibiotic pastes negatively impair dental pulp stem cell (DPSC) survival [2] and growth factors release from dentine [3], key aspects in the regenerative process. In light of this, a more cell-friendly disinfection strategy through the use of antibiotic-containing polymer scaffolds has been recently established [4], predicated on the fact that controlling the antibiotic(s) dose and release rate will lead to enhanced DPSC function. Here we report on the effects of dentine treatment with novel triple-antibiotic polymer scaffolds (TAPs) on human DPSC adhesion and proliferation.
Materials and Methods: Dentine slices (10×5×0.6 mm3) obtained from human mandibular incisors were irrigated with 17% EDTA, washed in saline solution, and UV sterilized. Electrospun TAPs (35 wt.% of antibiotics relative to the total polymer weight) were processed via electrospinning by adding metronidazole, ciprofloxacin, and minocycline (Sigma, St. Louis, MO) into a polydioxanone (PDS, Ethicon, Somerville, NJ) solution. PDS scaffolds with no antibiotics were also prepared. The TAP (50mg of each antibiotic), TAPs or antibiotic-free PDS scaffolds were placed in direct contact with the dentine surface for 7 days (n=4/group). The specimens were further rinsed with saline, followed by EDTA and a final rinse with saline. Human DPSCs (AllCells, Alameda, CA) at the third passage were seeded (1x104) on the dentine slices. Actin staining was done to evaluate cell adhesion at days 1 and 3 by confocal laser scanning microscopy (CLSM, Leica Microsystems Inc, Buffalo Grove, IL) and scanning electron microscopy (SEM, JSM-5310LV, JEOL, Tokyo). Cell proliferation was assessed at days 1, 3 and 7 using MTS assay (Promega, Madison, WI).
Results and Discussion: DPSCs showed enhanced cell adhesion and spreading on dentine slices treated with TAPs when compared to the TAP (Figure 1). Cell spreading was comparable for dentine treated with PDS and TAPs, which was similar to the control group (saline immersion). No evidence of cell spreading was noted on dentine treated with TAP. DPSCs proliferation rate was similar at Day 1 and 3 for dentine slices treated with PDS, TAP or TAPs. However, at Day 7 cell proliferation on dentine treated with the TAPs was significantly (9-fold; p<0.001) higher than TAP.
Conclusion: Taken together, the results demonstrate that the dentine slices treated with TAPs support DPSC adhesion and proliferation similar to the control. Conversely, the dentine slice with TAP alone did not support DPSC attachment and growth. Thus, the proposed triple-antibiotic polymer-based scaffold (TAPs) holds significant promise as a scaffold for dental pulp tissue engineering not only due to the proven antimicrobial properties but also due to the ability to support cell growth.
National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) (Grant #DE023552); American Association of Endodontists Foundation; Start up funds from Indiana University School of Dentistry; International Development Funds (IDF) Grant from Indiana University Purdue University (IUPUI/OVCR)
References:
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