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Abstract Background Application of pulp regenerative cell therapy for mature teeth with periapical lesions is a critical clinical challenge. The bacterial infection in inaccessible location within the root canal system and in the periapical lesions could cause resistance and impediment, leading to limitations in successful therapy. Thus, the aim of this study was to examine the effect of residual bacteria on the outcome of pulp regeneration in mature teeth with apical periodontitis in dogs. Methods Periapical lesions were induced in 32 root canals of 4 dogs in two different models in severities, model A and model B. Model A (moderate infection): the canal exposed to the oral cavity for 2 weeks and then closed for 2 weeks. Model B (severe infection): the canal exposed to the oral cavity for 2 months and then closed for 5 months. All root canals were irrigated with 6% sodium hypochlorite, and 3% EDTA and further with 0.015% levofloxacin-containing nanobubbles, which was also used as an intracanal medicament. The aseptic conditions were examined by bacterial anaerobic culture and/or PCR analyses. The root canal treatment was repeated several times, and allogeneic dental pulp stem cells were transplanted into the root canals. The radiographic evaluation of periapical lesions was performed by cone-beam computed tomography before the first treatment, just after cell transplantation, and after 2 months and 6 months in both model A, model B, respectively. The animals were then sacrificed and the jaw blocks were harvested for histological and histobacteriological evaluations of pulp regeneration and periapical tissue healing. Furthermore, the DiI-labelled DPSCs were transplanted into the root canals after complete disinfection (n = 4) or without root canal treatment (n = 4) in the apical periodontitis model (model A) in one dog, and cell localization was compared 72 h after transplantation. Results In 8 out of 12 canals from model A, and 10 out of 15 canals from model B, pulp regeneration with good vascularization, innervation, and a significant reduction in the radiolucent area of the periapical lesions were observed. However, in the other 4 canals and 5 canals from model A and model B, respectively, no pulp tissue was regenerated, and inflammation in the periapical tissue, and external resorption or healed external resorption were detected. The presence of residual bacteria in the periapical tissues and severe inflammation were significantly associated with inhibition of regenerated pulp tissue in these 9 unsuccessful canals (P

Abstract Background Clinical studies have demonstrated that dental pulp stem cells isolated from permanent teeth (PT-DPSCs) are safe and efficacious for complete pulp regeneration in mature pulpectomized permanent teeth with complete apical closure. Moreover, dental pulp stem cells from deciduous teeth (DT-DPSCs) have also been shown to be useful for pulp regenerative cell therapy of injured immature permanent teeth. However, direct comparisons of the pulp regenerative potential of DT-DPSCs and PT-DPSCs from the same individual have not been performed. This study aimed to compare the differences in stem cell properties and pulp regenerative potential of DT-DPSCs and PT-DPSCs of identical origin. Methods DT-DPSCs and PT-DPSCs were isolated from the same individual dogs at 4 months and 9 months of age, respectively. The expression of cell surface antigen markers, proliferation and migration activities, and gene expression of stem cell markers, angiogenic/neurotrophic factors and senescence markers were compared. The effects of conditioned medium (CM) derived from these cells on cellular proliferation, migration, angiogenesis, neurite outgrowth and immunosuppression were also compared. Autologous transplantation of DT-DPSCs or PT-DPSCs together with G-CSF was performed to treat pulpectomized teeth in individual dogs. The vascularization and reinnervation of the regenerated pulp tissues were qualitatively and quantitatively compared between groups by histomorphometric analyses. Results The rates of positive CXCR4 and G-CSFR expression in DT-DPSCs were significantly higher than those in PT-DPSCs. DT-DPSCs migrated at a higher rate with/without G-CSF and exhibited increased expression of the stem cell markers Oct3/4 and CXCR4 and the angiogenic factor VEGF and decreased expression of the senescence marker p16 than PT-DPSCs. DT-DPSC-derived CM promoted increased cell proliferation, migration with G-CSF, and angiogenesis compared with PT-DPSC-derived CM; however, no difference was observed in neurite outgrowth or immunosuppression. The regenerated pulp tissues in the pulpectomized teeth were quantitatively and qualitatively similar between the DT-DPSCs and PT-DPSCs transplant groups. Conclusions These results demonstrated that DT-DPSCs could be a potential clinical alternative to PT-DPSCs for pulp regenerative therapy. DT-DPSCs can be preserved in an individual cell bank and used for potential future pulp regenerative therapy before the supply of an individual’s own sound discarded teeth has been exhausted.

Abstract Background Dental pulp stem cells (DPSCs) have been developed as a potential source of mesenchymal stem cells (MSCs) for regeneration of dental pulp and other tissues. However, further strategies to isolate highly functional DPSCs beyond the colony-forming methods are required. We have demonstrated the safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. The device for isolation of MDPSCs, however, is not cost-effective and requires a prolonged cell culture period. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness. Therefore, in this investigation, we attempted to improve the clinical utility of DPSCs by hypoxia-preconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry. Methods Colony-forming DPSCs were isolated and preconditioned with hypoxia in a stable closed cultured system and compared with MDPSCs isolated from the individual dog teeth. We examined the proliferation rate, migration potential, anti-apoptotic activity, and gene expression of the stem cell markers and angiogenic/neurotrophic factors. Trophic effects of the conditioned medium (CM) were also evaluated. In addition, the expression of immunomodulatory molecules upon stimulation with IFN-γ was investigated. The pulp regenerative potential and transplantation safety of hpDPSCs were further assessed in pulpectomized teeth in dogs by histological and immunohistochemical analyses and by chemistry of the blood and urine tests. Results hpDPSCs demonstrated higher proliferation rate and expression of a major regulator of oxygen homeostasis, HIF-1α, and a stem cell marker, CXCR-4. The direct migratory activity of hpDPSCs in response to G-CSF was significantly higher than MDPSCs. The CM of hpDPSCs stimulated neurite extension. However, there were no changes in angiogenic, migration, and anti-apoptotic activities compared with the CM of MDPSCs. The expression of immunomodulatory gene, PTGE was significantly upregulated by IFN gamma in hpDPSCs compared with MDPSCs. However, no difference in nitric oxide was observed. The regenerated pulp tissue was quantitatively and qualitatively similar in hpDPSC transplants compared with MDPSC transplants in dog teeth. There was no evidence of toxicity or adverse events of the hpDPSC transplantation. Conclusions These results demonstrated that the efficacy of hpDPSCs for pulp regeneration was identical, although hpDPSCs improved stem cell properties compared to MDPSCs, suggesting their potential clinical utility for pulp regeneration.

Abstract Background We recently demonstrated that autologous transplantation of mobilized dental pulp stem cells (MDPSCs) was a safe and efficacious potential therapy for total pulp regeneration in a clinical study. The autologous MDPSCs, however, have some limitations to overcome, such as limited availability of discarded teeth from older patients. In the present study, we investigated whether MDPSCs can be used for allogeneic applications to expand their therapeutic use. Methods Analysis of dog leukocyte antigen (DLA) was performed using polymerase chain reaction from blood. Canine allogeneic MDPSCs with the matched and mismatched DLA were transplanted with granulocyte-colony stimulating factor in collagen into pulpectomized teeth respectively (n = 7, each). Results were evaluated by hematoxylin and eosin staining, Masson trichrome staining, PGP9.5 immunostaining, and BS-1 lectin immunostaining performed 12 weeks after transplantation. The MDPSCs of the same DLA used in the first transplantation were further transplanted into another pulpectomized tooth and evaluated 12 weeks after transplantation. Results There was no evidence of toxicity or adverse events of the allogeneic transplantation of the MDPSCs with the mismatched DLA. No adverse event of dual transplantation of the MDPSCs with the matched and mismatched DLA was observed. Regenerated pulp tissues including neovascularization and neuronal extension were quantitatively and qualitatively similar at 12 weeks in both matched and mismatched DLA transplants. Regenerated pulp tissue was similarly observed in the dual transplantation as in the single transplantation of MDPSCs both with the matched and mismatched DLA. Conclusions Dual allogeneic transplantation of MDPSCs with the mismatched DLA is a safe and efficacious method for total pulp regeneration.