Center (UTHSC) based on exempt protocols approved by the respective Medical Institutional Review Board (BU: #H-28882; and UTHSC:#12-01937-XM). • Consent for publication: This manuscript has been approved by all authors and is solely the work of the authors named. • Availability of data and material: All data and information of the materials relevant to this project are available upon request. Cell lines established and used in the project will be available upon request.

IntroductionInduced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we have generated iPSCs from human dental stem cells carrying transgene vectors. These exogenous transgenes may affect iPSC behaviors and limit their clinical applications. The purpose of this study was to establish transgene-free iPSCs (TF-iPSCs) reprogrammed from human stem cells of apical papilla (SCAP) and determine their neurogenic potential.MethodsA single lentiviral 'stem cell cassette' flanked by the loxP site (hSTEMCCA-loxP), encoding four human reprogramming factors, OCT4, SOX2, KLF4, and c-MYC, was used to reprogram human SCAP into iPSCs. Generated iPSCs were transfected with plasmid pHAGE2-EF1α-Cre-IRES-PuroR and selected with puromycin for the TF-iPSC subclones. PCR was performed to confirm the excision of hSTEMCCA. TF-iPSC clones did not resist to puromycin treatment indicating no pHAGE2-EF1α-Cre-IRES-PuroR integration into the genome. In vitro and in vivo analyses of their pluripotency were performed. Embryoid body-mediated neural differentiation was undertaken to verify their neurogenic potential.ResultsTF-SCAP iPSCs were generated via a hSTEMCCA-loxP/Cre system. PCR of genomic DNA confirmed transgene excision and puromycin treatment verified the lack of pHAGE2-EF1α-Cre-IRES-PuroR integration. Transplantation of the TF-iPSCs into immunodeficient mice gave rise to teratomas containing tissues representing the three germ layers -- ectoderm (neural rosettes), mesoderm (cartilage and bone tissues) and endoderm (glandular epithelial tissues). Embryonic stem cell-associated markers TRA-1-60, TRA-2-49 and OCT4 remained positive after transgene excision. After neurogenic differentiation, cells showed neural-like morphology expressing neural markers nestin, βIII-tubulin, NFM, NSE, NeuN, GRM1, NR1 and CNPase.ConclusionsTF-SCAP iPSCs reprogrammed from SCAP can be generated and they may be a good cell source for neurogenesis.

Regenerative endodontic procedures (REPs) is a biologic-based treatment modality for immature permanent teeth diagnosed with pulp necrosis. The ultimate objective of REPs is to regenerate the pulp-dentin complex, extend the tooth longevity and restore the normal function. Scientific evidence has demonstrated the efficacy of REPs in promotion of root development through case reports, case series, cohort studies, and randomized controlled studies. However, variations in clinical protocols for REPs exist due to the empirical nature of the original protocols and rapid advancements in the research field of regenerative endodontics. The heterogeneity in protocols may cause confusion among dental practitioners, thus guidelines and considerations of REPs should be explicated. This expert consensus mainly discusses the biological foundation, the available clinical protocols and current status of REPs in treating immature teeth with pulp necrosis, as well as the main complications of this treatment, aiming at refining the clinical management of REPs in accordance with the progress of basic researches and clinical studies, suggesting REPs may become a more consistently evidence-based option in dental treatment.

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Induced pluripotent stem cells (iPSCs) give rise to neural stem/progenitor cells, serving as a good source for neural regeneration. Here, we established transgene-free (TF) iPSCs from dental stem cells (DSCs) and determined their capacity to differentiate into functional neurons in vitro. Generated TF iPSCs from stem cells of apical papilla and dental pulp stem cells underwent two methods-embryoid body-mediated and direct induction, to guide TF-DSC iPSCs along with H9 or H9 Syn-GFP (human embryonic stem cells) into functional neurons in vitro. Using the embryoid body-mediated method, early stage neural markers PAX6, SOX1, and nestin were detected by immunocytofluorescence or reverse transcription-real time polymerase chain reaction (RT-qPCR). At late stage of neural induction measured at Weeks 7 and 9, the expression levels of neuron-specific markers Nav1.6, Kv1.4, Kv4.2, synapsin, SNAP25, PSD95, GAD67, GAP43, and NSE varied between stem cells of apical papilla iPSCs and H9. For direct induction method, iPSCs were directly induced into neural stem/progenitor cells and guided to become neuron-like cells. The direct method, while simpler, showed cell detachment and death during the differentiation process. At early stage, PAX6, SOX1 and nestin were detected. At late stage of differentiation, all five genes tested, nestin, βIII-tubulin, neurofilament medium chain, GFAP, and Nav, were positive in many cells in cultures. Both differentiation methods led to neuron-like cells in cultures exhibiting sodium and potassium currents, action potential, or spontaneous excitatory postsynaptic potential. Thus, TF-DSC iPSCs are capable of undergoing guided neurogenic differentiation into functional neurons in vitro, thereby may serve as a cell source for neural regeneration.

The aim of this study was to compare the efficiency of four final irrigation protocols in smear layer removal and bacterial inhibition in root canal systems. Thirty roots inoculated with Enterococcus faecalis were prepared with ProTaper Universal files. The teeth were disinfected by conventional needle irrigation, sonic agitation using the EndoActivator device, passive ultrasonic irrigation, or an M3 Max file. Teeth with no root canal preparation served as blank controls for the establishment of the infection baseline. Teeth with preparation but no final irrigation served as a post-instrumentation baseline. After the final irrigation, the teeth were sectioned in half. One half of each tooth was examined by scanning electron microscopy (SEM) to assess smear layer removal using a five-point scale. The other half was examined by confocal laser scanning microscopy (CLSM) using the LIVE/DEAD BackLight bacterial viability kit to evaluate the depth of bacterial survival in dentinal tubules. SEM analysis revealed no significant difference in smear layer removal throughout the whole canal among the EA, PUI, and M3 Max groups (P > 0.05). CLSM revealed that PUI achieved the greatest bacterial inhibition depth in the coronal ((174.27 ± 31.63) μm), middle ((160.94 ± 37.77) μm), and apical ((119.53 ± 28.49) μm) thirds of the canal (all P < 0.05 vs. other groups). According to this comprehensive SEM and CLSM evaluation, PUI appears to have the best infection control ability in root canal systems.