Responses of oral-microflora-exposed dental pulp to capping with a triple antibiotic paste or calcium hydroxide cement in mouse molars

Angela Quispe-Salcedo; Takuichi Sato; Junko Matsuyama; Hiroko Ida-Yonemochi; Hayato Ohshima

doi:10.1016/j.reth.2020.10.001

Responses of oral-microflora-exposed dental pulp to capping with a triple antibiotic paste or calcium hydroxide cement in mouse molars

Regen Ther. 2020 Oct 29:15:216-225. doi: 10.1016/j.reth.2020.10.001. eCollection 2020 Dec.

Authors

Angela Quispe-Salcedo^{1

2}, Takuichi Sato³, Junko Matsuyama^{4

5}, Hiroko Ida-Yonemochi¹, Hayato Ohshima¹

Affiliations

¹ Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan.
² School of Stomatology, Faculty of Health Science, Universidad Cientifica del Sur, Av. Paseo de la República 5544, Miraflores, 15074, Lima, Peru.
³ Division of Clinical Chemistry, Department of Medical Technology, Niigata University Graduate School of Health Sciences, 2-746 Asahimachi-dori, Chuo-ku, Niigata, 951-8518, Japan.
⁴ Division of Pediatric Dentistry, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan.
⁵ Niigata City Dental Health and Welfare Center, 3-3-11 Shichikuyama, Chuo-ku, Niigata, 950-0914, Japan.

Abstract

Introduction: Responses of oral-microflora-exposed dental pulp to a triple antibiotic paste (TAP), a mixture of ciprofloxacin, metronidazole, and minocycline in ointment with macrogol and propylene glycol, remain to be fully clarified at the cellular level. This study aimed to elucidate responses of oral-microflora-exposed dental pulp to capping with TAP in mouse molars.

Methods: A cavity was prepared on the first molars of 6-week-old mice to expose the dental pulp for 24 h. The exposed pulp was capped with TAP (TAP group) or calcium hydroxide cement (CH group), in addition to the combination of macrogol (M) and propylene glycol (P) (MP, control group), followed by a glass ionomer cement filling. The samples were collected at intervals of 1, 2, and 3 weeks, and immunohistochemistry for nestin and Ki-67 and deoxyuride-5'-triphosphate biotin nick end labeling (TUNEL) assay were performed in addition to quantitative real-time polymerase chain reaction (qRT-PCR) analyses.

Results: The highest occurrence rate of pulp necrosis was found in the control group followed by the CH group at Weeks 2 and 3, whereas the highest occurrence rate of healed areas in the dental pulp was observed in the TAP group at each time point. Tertiary dentin formation was first observed in the dental pulp of the TAP group at Week 2. In contrast, bone-like and/or fibrous tissues were frequently observed in the CH group. qRT-PCR analyses clarified that TAP activated the stem and dendritic cells at Weeks 1 and 2, respectively.

Conclusions: The use of TAP as a pulp-capping agent improved the healing process of oral-microflora-exposed dental pulp in mouse molars.

Keywords: ANOVA, One-way analysis of variance; AZAN, Azocarmine and aniline blue; Anti-bacterial agents; BMPs, Bone morphogenetic proteins; Birc5, Baculoviral IAP Repeat Containing 5; CH, Calcium hydroxide; Cell differentiation; Cell proliferation; Ct, Cycle threshold; DAP, Double antibiotic paste; DCs, Dendritic cells; DNA, Deoxyribonucleic acid; DPC, Direct pulp capping; DPSCs, Dental pulp stem cells; Dental cavity preparation; Dental pulp; FGFs, Fibroblast growth factors; GM-CSF, Granulocyte-macrophage colony-stimulating factor; H2O2, Hydrogen peroxide; HE, Hematoxylin-eosin; HLA-DR-immunopositive cells, Human Leukocyte Antigen – DR isotype-immunopositive cells; M, Macrogol; MHC, Major histocompatibility complex; MP, Macrogol (M) mixed with propylene glycol (P); MSCs, Mesenchymal stem cells; MTA, Mineral trioxide aggregate; Mice (crlj:CD1); Oct 3/4 A, Octamer binding transcription factor 3/4 A; Oct 3/4 B, Octamer binding transcription factor 3/4 B; P, Propylene glycol; PBS, Phosphate-buffered saline; Pcna, Proliferating cell nuclear antigen; REP, Regenerative endodontic procedures; RNA, Ribonucleic acid; RT, Reverse transcription; SCAP, Stem cells of the apical papilla; Sox 10, SRY-related HMG-box 10; TAC, Triple antibiotic combination (a mixture of metronidazole, ciprofloxacin, and minocycline); TAC-P, Triple antibiotic combination and propylene glycol; TAP, Triple antibiotic paste; TAS, Triple antibiotic solution; TGFβ, Transforming growth factor β; TUNEL assay, Terminal deoxynucleotidyl transferase dUTP nick end labeling assay; Tris–HCl buffer, Tris (hydroxymethyl) aminomethane (THAM) hydrochloride buffer; Yap1, Yes-associated protein 1; cDNA, Complementary deoxyribonucleic acid; mRNA, Messenger ribonucleic acid; mTAP, Modified triple antibiotic paste; qRT-PCR, Quantitative real-time polymerase chain reaction; αTCP, Alpha tricalcium phosphate; β-actin, Beta-actin.