[P3-2-10] Dental pulp cell transplantation in combination with regenerative endodontic procedures promote dentin matrix formation in mouse molars
キーワード：Regenerative endodontic procedures、dental pulp regeneration、dental pulp cells
Purpose: We hypothesized that performing regenerative endodontic procedures (REP), which consist of root canal disinfection and provoking intracanal bleeding, in combination with dental pulp cell (DPC) transplantation, would promote dentin matrix formation in mature molars. Therefore, the aim of this study is to develop a mouse model of REP in combination with DPC transplantation and assess dental pulp regeneration in mature teeth.
Materials & Methods: REP was performed in mandibular first molars of 10-week-old C57BL/6 mice by instrumentation of the mesial and distal canals with #6, #7 and #8 K files and irrigation with NaClO and EDTA. Subsequently, intracanal bleeding was provoked and the canals were left untreated (REP group) or immediately transplanted with DPCs obtained from neonatal GFP mice (REP +DPC group). The pulp cavity was filled with MTA and sealed with composite resin. After 4 weeks, the regenerated tissue was evaluated by micro-CT (μCT), H&E staining and immunohistochemical analyses with nestin (odontoblast marker), CD31 (endothelial cell marker) and GFP antibodies to identify transplanted DPCs. Prior to transplantation, DPCs were stained with vimentin and cytokeratin antibodies, to examine mesenchymal and epithelial cells, respectively. Also, the cells were stained with KLF4 to confirm pluripotency.
Results: DPCs were uniformly stained for KLF4. In addition, 50% of the cells were vimentin+, while less than 3% were cytokeratin+. μ-CT analysis showed that after 4 weeks of REP treatment, the teeth showed no signs of periapical lesions. H&E-stained tissue sections showed pulp-like tissue formation in both the REP and REP+DPCs groups. The regenerated pulp-like tissue consisted of a cellular matrix, with vessel-like structures, surrounded by an eosin-stained acellular matrix that resembled hard tissue. However, the cellular matrix was larger in the REP+DPC group than in the REP group. Furthermore, nestin+ cells (odontoblasts) were found only in the REP+DPC group, and these cells co-expressed GFP. Moreover, the number of CD31+ vessels and their diameter were greater in the REP+DPC group than in the REP group.
Conclusion: DPC transplantation may improve outcomes of REP by inducing the formation of odontoblast-like cells and greater vasculogenesis.
Materials & Methods: REP was performed in mandibular first molars of 10-week-old C57BL/6 mice by instrumentation of the mesial and distal canals with #6, #7 and #8 K files and irrigation with NaClO and EDTA. Subsequently, intracanal bleeding was provoked and the canals were left untreated (REP group) or immediately transplanted with DPCs obtained from neonatal GFP mice (REP +DPC group). The pulp cavity was filled with MTA and sealed with composite resin. After 4 weeks, the regenerated tissue was evaluated by micro-CT (μCT), H&E staining and immunohistochemical analyses with nestin (odontoblast marker), CD31 (endothelial cell marker) and GFP antibodies to identify transplanted DPCs. Prior to transplantation, DPCs were stained with vimentin and cytokeratin antibodies, to examine mesenchymal and epithelial cells, respectively. Also, the cells were stained with KLF4 to confirm pluripotency.
Results: DPCs were uniformly stained for KLF4. In addition, 50% of the cells were vimentin+, while less than 3% were cytokeratin+. μ-CT analysis showed that after 4 weeks of REP treatment, the teeth showed no signs of periapical lesions. H&E-stained tissue sections showed pulp-like tissue formation in both the REP and REP+DPCs groups. The regenerated pulp-like tissue consisted of a cellular matrix, with vessel-like structures, surrounded by an eosin-stained acellular matrix that resembled hard tissue. However, the cellular matrix was larger in the REP+DPC group than in the REP group. Furthermore, nestin+ cells (odontoblasts) were found only in the REP+DPC group, and these cells co-expressed GFP. Moreover, the number of CD31+ vessels and their diameter were greater in the REP+DPC group than in the REP group.
Conclusion: DPC transplantation may improve outcomes of REP by inducing the formation of odontoblast-like cells and greater vasculogenesis.