Abstract
BackgroundDental 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.MethodsColony-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.ResultshpDPSCs 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.ConclusionsThese 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.
Highlights
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
The direct migratory activity of hypoxiapreconditioned DPSCs (hpDPSCs) in response to granulocyte colonystimulating factor (G-CSF) was significantly higher than mobilized DPSCs (MDPSCs)
There were no changes in angiogenic, migration, and anti-apoptotic activities compared with the conditioned medium (CM) of MDPSCs
Summary
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. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness In this investigation, we attempted to improve the clinical utility of DPSCs by hypoxiapreconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry. The isolation methods by the flow cytometer or magnetic cell selection system device using the stem cell marker CD105 were not cost-effective in safety and efficacy for manufacturing clinical grade DPSCs. we have developed a novel isolation method based on ability of DPSC subfractions to mobilized by granulocyte colonystimulating factor (G-CSF) [9]. The cost-effective method and safety of the isolation and processing of good manufacturing practice (GMP) grade DPSC subsets with high-regeneration potential remains a challenge, and we have addressed this in the present investigation
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