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Background Teeth with dens evaginatus (DE) are more commonly observed in Western countries than previously. This is due to the increase in populations of patients of Asian origin, in whom DE is more common than in people of European origin. The interest in DE has also increased with the introduction of a procedure called regenerative endodontics. Case Description A narrative review of treatment options for teeth with DE is presented, based on pulpal conditions and maturity of the teeth. Practical Implications Early recognition of teeth with DE allows for treatment choices that generally lead to good outcomes and can aid in preserving developing teeth in young patients.

Endodontics has made significant progress in regenerative approaches in recent years, thanks to advances in biologically based procedures or regenerative endodontic therapy (RET). In recent years, our profession has witnessed a clear conceptual shift in this therapy. RET was initially based on a blood clot induced by apical bleeding without harvesting the patient’s cells or cell-free RET. Later, the RET encompassed the three principles of tissue engineering, stromal/stem cells, scaffolds, and growth factors, aiming for the regeneration of a functional dentin pulp complex. The regenerated dental pulp will recover the protective mechanisms including innate immunity, tertiary dentin formation, and pain sensitivity. This comprehensive review covers the basic knowledge and practical information for translational applications of stem cell-based RET and tissue engineering procedures for the regeneration of dental pulp. It will also provide overall information on the emerging technologies in biological and synthetic matrices, biomaterials, and signaling molecules, recent advances in stem cell therapy, and updated experimental results. This review brings useful and timely clinical evidence for practitioners to understand the challenges faced for a successful cell-based RET and the importance of preserving or reestablishing tooth vitality. The clinical translation of these current bioengineering approaches will undoubtedly be beneficial to the future practice of endodontics.

This study investigated a colloidal microgel for angiogenic and odontogenic differentiation of cells in the presence of cell-derived extracellular matrix (ECM) proteins using a 3-dimensional culture model.Viscoelastic properties of human dental pulp were determined to understand the native ECM environment. ECM proteins were extracted from dental pulp stem cell (DPSC) cultures, and MaxGel (Millipore Sigma, Burlington, MA) was used as a commercially available ECM protein. DPSCs were incubated in colloidal microgels in the presence of ECM proteins or gelatin methacryloyl (GelMA) as a bulk hydrogel (n = 9/group). The viability and odontogenic differentiation of DPSCs within hydrogels was determined using viability assays, mineralization staining, calcium and alkaline phosphatase assays, and quantitative polymerase chain reaction for odontogenic gene expression. Angiogenic properties of endothelial cells were determined using tubule formation assays and quantitative polymerase chain reaction to detect angiogenic gene expression.Dental pulp had a higher elastic modulus than the viscous modulus, showing a solidlike response similar to hydrogels. DPSC-derived ECM showed higher collagen and GAG than MaxGel (P .05). The viability of DPSCs was similar in colloidal microgels, whereas higher cell viability, calcium deposition, and alkaline phosphatase activity were observed in GelMA (P .05). Colloidal microgels allowed tubule-like structures by endothelial cells, whereas no tubular formation was observed in GelMA. DPSC-derived ECM in colloidal microgel up-regulated odontogenic gene expression, whereas MaxGel up-regulated angiogenic gene expression (P .05).Colloidal microgels allowed cellular organization that can improve penetration and nutritional supply in a full-length root canal system. The bioactivity of cell-derived ECM proteins can be modified depending on the external stimulus.

Objective : This study explored if human primary mesenchymal stem cells (MSCs), derived from two donors and cultivated in a medium made with intentionally treated water, would exhibit more growth and pluripotency than MSCs from the same source but grown in untreated (control) water. Design : To create the treated water, three Buddhist monks directed their attention toward commercially bottled water while holding the intention that the water would enhance the growth of MSCs. Under double-blind conditions, cell culture growth mediums were prepared with the treated and untreated water, which was in turn used to grow the primary MSCs. Primary cells obtained from two donors were designated as Cells #1 and Cells #2. The prediction was that treated water would result in increased cell proliferation, that more cells would enter the cell cycle growth phase, and that there would be increased expression of genes (NANOG, OCT4 and SOX2) associated with improved cell growth and decreased expression of genes (p16, p21, and p53) associated with a decline in cell growth. The improved growth hypothesis was directional, thus one-tailed p-values were used to evaluate the results. Results : Proliferation averaged across Cells #1 and #2 showed overall increased growth in treated as compared to control water (p = 0.0008). Cells #1 and #2 considered separately had differences in the same direction but only Cells #2 showed a significant difference on day 6 (p = 0.01). For cell cycle, there was a significantly greater percentage of Cells #2 in the S interphase with treated vs. control water (p = 0.04). For the gene expression analysis, when considering the average across the two donor cells, only the NANOG gene expression was in the predicted direction (p = 0.01); by contrast, the p16 gene expression was significantly opposite to the predicted direction (p = 0.005, one-tailed, post-hoc). For Cells #1 considered separately, no differences were significant except for p16, which resulted in an effect opposite to the predicted outcome (p = 0.05). For Cells #2, three genes were significantly in the predicted directions: NANOG (p = 0.0008), OCT4 (p = 0.005), and P53 (p = 0.05); p16 was significantly opposite to the prediction (p = 0.001). Conclusion : Intentionally treated water appeared to have some biological effects on the growth, pluripotency and senescence of human MSCs. This was especially the case in one of the two donor cells tested, but the effects were not consistently in the predicted direction. As an exploratory study, caution is warranted in interpreting these outcomes, and adjustment for multiple testing would likely reduce some of the weaker effects to nonsignificant. But given the double-blind protocol, as well as several more significant outcomes in the predicted directions, further research is warranted.

2-Aminoethyl methacrylate (AEMA)–modified hyaluronic acid (HA) hydrogels with tunable swelling, degradation, and rheological properties were developed to maintain the native spherical and stemness of human dental pulp stem cells (DPSCs). Two molecular weights (MW) of HA, 18 and 270 kDa, and 25% degree of AEMA substitution were used to synthesize the HA macromers and hydrogels. The HA macromers were characterized by 1H NMR, and the AEMA-HA hydrogels were synthesized by UV polymerization. The swelling, hydrolytic degradation, and rheological properties of the hydrogels strongly depended on the MW of HA. The degradation rate decreased and the storage modulus increased with the increase in the MW of HA. The storage modulus of the hydrogels decreased with degradation of the hydrogels. The hydrogels were not cytotoxic to DPSCs evidenced by MTT assay, Hoechst 33342 live cell staining, and propidium iodide dead cell staining. The hydrogels maintained the native spherical morphology and stemness of DPSCs when the hydrogels’ storage moduli were in the ranges of 486–681 Pa. In contrast, DPSCs grown on 2D cell culture substrates showed a flat and elongated shape and could not maintain their native spherical shape. The hydrogels increased the expression of the stemness including NANOG and SOX2 of DPSCs (3 and 24 folds, respectively) when their storage modulus was 681.41 ± 92.81 Pa in comparison with DPSCs grown on 2D cell culture plates. The designed AEMA-HA hydrogels mimic the in vivo 3D environment to grow DPSCs. Thus, they appear to have great potential for clinical applications in regenerative medicine and other therapeutic remedies after further optimization in the future. Regenerative medicine is a translational approach that offers the possibility to replace or heal the diseased or damaged tissues caused by trauma, age, infection, cancer, or inherited defects. Such reinstatement of the functionality or healing of the damaged cells or tissues often includes the combination of numerous strategies including the use of biomaterials/scaffolds, stem cells, growth factors, and small molecules. In this study, we used hyaluronic acid (HA), a naturally occurring polysaccharide biopolymer commonly found in the extracellular matrix of connective tissues, to synthesize 3D hydrogel scaffolds for growing human dental pulp stem cells (DPSCs). We evaluated the effects of the molecular weight of HA, and the swelling, degradation, and rheological properties of the hydrogels on maintaining the native spherical and stemness of DPSCs for future therapeutic applications including regenerative medicine.

Rapid progress has been made in the last decade related to stem cell-mediated pulp–dentin regeneration, from characterization of dental pulp stem cells (DPSCs) to the first-ever reported clinical case in humans. However, many challenges still need to be addressed before such technology can become a common clinical practice; therefore, further rigorous research is needed. Animal study models are very important to test new ideas, concepts, and technologies. This review summarizes and discusses several key animal models that have been utilized to investigate pulp–dentin regeneration. From a tissue regeneration perspective, we categorize the animal model by the location where the regenerated pulp tissue is formed: ectopic, semiorthotopic, and orthotopic. Several animal species are discussed, including mouse, ferret, dog, and miniswine. Mouse is used for ectopic pulp–dentin regeneration in the dorsum subcutaneous space. A commonly tested approach is hydroxyapatite/tricalcium phosphate (HA-TCP) granules model used to observe ectopic pulp–dentin complex formation. The semiorthotopic model includes tooth slices or fragments with which de novo pulp regeneration in a root canal space can be tested in the mouse subcutaneous space. For orthotopic pulp regeneration, the canine teeth of ferrets are large enough for such purposes. As nonprimate large animal models, dog and miniswine teeth have many aspects quite similar to those of humans, allowing researchers to perform experiments that mimic clinical conditions in humans. The protocols established and the data obtained from large animal studies may directly relate to and apply to future human studies. Complete orthotopic pulp regeneration has been demonstrated in dogs and miniswine. The use of allogeneic and subpopulations of DPSCs for pulp regeneration, and testing of the periapical disease model and aging model have been performed in miniswine or dogs. In sum, all these animal models will help address challenges that still face pulp regeneration in humans. We need to thoroughly utilize these models to test new ideas, technologies, and strategies before reliable and predictable clinical protocols can be established for human clinical trials or treatment. IMPACT STATEMENT: Animal models are essential for tissue regeneration studies. This review summarizes and discusses the small and large animal models, including mouse, ferret, dog, and miniswine that have been utilized to experiment and to demonstrate stem cell-mediated dental pulp tissue regeneration. We describe the models based on the location where the tissue regeneration is tested—either ectopic, semiorthotopic, or orthotopic. Developing and utilizing optimal animal models for both mechanistic and translational studies of pulp regeneration are of critical importance to advance this field.

Craniofacial and dental tissues are unique and contain specialized cell types. The embryonic origins and characteristics of craniofacial bone are different from long bone. Craniofacial tissues are derived from neural crest cells (NCCs). The evidence has shown that MSCs derived from craniofacial tissue behave differently from those of other tissue origin. Thus, using craniofacial MSCs appears to be a better cell source for craniofacial tissue regeneration. While adult stem cells offer various clinical benefits for tissue regeneration, their key drawback is the limited lifespan and differentiation potential for certain applications. Induced pluripotent stem cells (iPSCs) can overcome these deficiencies. Oral and dental stem cells are also a good source of cells to generate iPSCs. Generated iPSCs either from dental and nondental cell sources have been investigated for their capacity in neurogenesis as well as for craniofacial tissue regeneration, including craniofacial bone regeneration, pulp regeneration, periodontal tissue regeneration, and whole tooth regeneration. Before iPSCs can provide significant clinical benefits for patients, issues revolving iPSC biology, their translation, and clinical advancement that pose various challenges need to be resolved.

There is no consensus on the true meaning of clinical regenerative endodontics, and there is confusion over the concept and the term. Commonly used terms include revitalization and revascularization. The clinical methods for endodontic revitalization procedures and the tissue engineering concept differ depending on whether there is exogenous delivery of cells - called cell therapy, or not. Here, in this review, the difference is clarified by emphasizing the correct terminology: cell-free versus cell-based regenerative endodontic therapy (CF-RET versus CB-RET). The revitalization procedures practised clinically do not fit into the modern tissue engineering concepts of pulp regeneration but can be categorized as CF-RET. The modern tissue engineering concept in pulp regeneration is a CB-RET, which so far is at the clinical trial stage. However, histological examination of teeth following regenerative endodontic treatments reveals healing with repair derived from stem cells that originate from the periodontal, bone and other tissues. The aim of regenerative endodontics is regeneration of the pulp-dentine complex. This review discusses why CF-RET is unlikely to regenerate a pulp-dentine complex with current protocols. The American Association of Endodontists and the European Society of Endodontology have not yet recommended autologous stem cell transplantation (CB-RERT) which aspires for regeneration. Therefore, an understanding of the concept, term, difficulties and differences in current protocols is important for the clinician. However, rather than being discouraged that ideal regeneration has not been achieved to date, repair can be an acceptable outcome in clinical regenerative endodontics as it has also been accepted in medicine. Repair should also be considered in the context that resolution of the clinical signs/symptoms of pulp necrosis/apical periodontitis is generally reliably obtained in clinical regenerative endodontics.

Stem cell-mediated regenerative endodontics has reached the human clinical trial phase; however, many issues still exist that prevent such technology to be a widely used clinical practice. These issues are not straightforward and are complicated. They should be because pulp regeneration is dealing with a small dead-end space. In addition, when regeneration is needed, the space is often heavily infected. The true standard of pulp regeneration should be everything except generation of some fibrous connective tissue and amorphous mineral deposit. As of now, we are still far short of reaching the standard of complete vascularized and innervated pulp regeneration with newly formed tubular dentin in all types of teeth. Thus, we need to go back to the bench and use established animal models or create new animal models to tackle those issues. This article will address several key issues including the possibility of pulp regeneration in small canals of molar teeth by enhancing the neovascularization, and whether the organized tubular dentin can be generated on the canal walls. Data from our semi-orthotopic tooth fragment mouse model have shown that complete pulp regeneration using dental pulp stem cells (DPSCs) in small canal has been inconsistent because of limited blood supply. This inconsistency is similar in our orthotopic miniature swine model, although in some cases vascularized pulp-like tissue can be formed throughout the canal space after DPSC transplantation. Furthermore, no tubular dentin was observed in the orthotopic pulp regeneration, despite the fact that DPSCs have the capacity to generate some tubular dentin-like structure in the hydroxyapatite/tricalcium phosphate-mediated ectopic pulp/dentin formation model in mice. Potential strategies to be tested to address these regeneration issues are discussed herein.

The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere-mediated or neurosphere-mediated methods to guide them toward neuronal lineages. Cells were subjected to RT-qPCR, immunocytofluorescence to detect the expression of neurogenic genes or electrophysiological analysis at final stage of maturation. We found that induced DPSCs and GMSCs overall appeared to be more neurogenic compared to other cells either morphologically or levels of neurogenic gene expression. Nonetheless, of all the neural induction methods employed, only one neurosphere-mediated method yielded electrophysiological properties of functional neurons. Under this method, cells expressed increased neural stem cell markers, nestin and SOX1, in the first phase of differentiation. Neuronal-like cells expressed βIII-tubulin, CNPase, GFAP, MAP-2, NFM, pan-Nav, GAD67, Nav1.6, NF1, NSE, PSD95, and synapsin after the second phase of differentiation to maturity. Electrophysiological experiments revealed that 8.3% of DPSC-derived neuronal cells and 21.2% of GMSC-derived neuronal cells displayed action potential, although no spontaneous excitatory/inhibitory postsynaptic action potential was observed. We conclude that DPSCs and GMSCs have the potential to become neuronal cells in vitro, therefore, these cells may be used as a source for neural regeneration.

Teeth with dens evaginatus (DE) are more commonly observed in Western countries than previously. This is due to the increase in populations of patients of Asian origin, in whom DE is more common than in people of European origin. The interest in DE has also increased with the introduction of a procedure called regenerative endodontics.A narrative review of treatment options for teeth with DE is presented, based on pulpal conditions and maturity of the teeth.Early recognition of teeth with DE allows for treatment choices that generally lead to good outcomes and can aid in preserving developing teeth in young patients.

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.

Introduction/Objective The tailored amorphous multi-porous (TAMP) material fabrication technology has led to a new class of bioactive materials possessing versatile characteristics. It has not been tested for dental applications. Thus, we aimed to assess its biocompatibility and ability to regenerate dental mineral tissue. Methods 30CaO-70SiO2 model TAMP discs were fabricated by a sol-gel method followed by in vitro biocompatibility testing with isolated human or mini-swine dental pulp stem cells (DPSCs). TAMP scaffolds were tested in vivo as a pulp exposure (pin-point, 1 mm, 2 mm, and entire pulp chamber roof) capping material in the molar teeth of mini-swine. Results The in vitro assays showed that DPSCs attached well onto the TAMP discs with comparable viability to those attached to culture plates. Pulp capping tests on mini-swine showed that after 4.5 months TAMP material was still present at the capping site, and mineral tissue (dentin bridge) had formed in all sizes of pulp exposure underneath the TAMP material. Conclusions TAMP calcium silicate is biocompatible with both human and swine DPSCs in vitro and with pulp in vivo, it may help regenerate the dentin bridge after pulp exposure.

This is to compare the volumes of irrigant apically extruded by five irrigation systems in an artificial socket model simulating clinical conditions. Twenty extracted human single-rooted teeth were enlarged to size 40/04 and then embedded in silicone impression material. The root canal space was irrigated with nominal 3% sodium hypochlorite (NaOCl) using standard needle irrigation (SNI) with a 30-gauge notched needle, EndoActivator (EA), XP Endo Finisher (XP Endo), EndoVac (EV), and photon-induced photoacoustic streaming (PIPS). Extruded NaOCl was collected, reacted with taurine to form taurine-monochloramine, and absorbance of taurine-monochloramine was measured at 252 nm using a spectrophotometer. The five irrigation systems were compared with repeated measures ANOVA and pairwise comparisons. The EV group had very low extrusion (mean ± SD = 0.12 ± 0.2 μL) and differed significantly from the other four groups (P ≤ 0.001). Larger volumes of irrigant were extruded in the other irrigation groups. There were no significant differences in the extruded volumes among the SNI (7.4 ± 3.4 μL), EA (7.0 ± 6.1 μL), and XP Endo (7.8 ± 4.1 μL) groups (P = 1). The PIPS group had the highest mean extruded volume (12.9 ± 6.8 μL) and differed significantly from SNI (P = 0.030), EV (P

In this study, we investigated the properties of nanobubble (NB) water and its effect on smear layer removal and strengthening the efficiency of disinfecting agents used in regenerative endodontic treatment.NB water was generated in a NB Generator. The NB size, concentration, and pH were measured. Porcine teeth were enlarged to size 60 by using hand-files and irrigated with either NB water or 17% EDTA or received no further irrigation. The ability of irrigants to remove the smear layer was evaluated by using a scanning electron microscope (9 roots/group). Other samples (6 roots/group) were subjected to Vickers hardness test to determine the dentin microhardness. Autofluorescent tetracycline mixed with distilled water or NB water was placed inside the root canal space of porcine teeth, and the depth of medicament penetration into the dentinal tubules was visualized by using fluorescent stereomicroscope (5 roots/group). For the disinfection experiment, human roots were prepared, autoclaved, and inoculated with Enterococcus faecalis for 3 weeks. Canals were then disinfected by (1) standard needle irrigation (SNI) with 5.25% NaOCl, (2) 5.25% NaOCl with ultrasonication (US), (3) 5.25% NaOCl + XP finisher (XP), (4) SNI with 1.5% NaOCl, or (5) SNI with 1.5% NaOCl in NB water (5 roots/group). Teeth were split open and stained with LIVE/DEAD BackLight and visualized by using confocal laser scanning microscope (CLSM) at the coronal, middle, and apical thirds of the canal. The ratio of dead/total bacteria in the dentinal tubules at various depth levels (50, 100, and 150 μm) was calculated.NB water was more effective in removing smear layer than 17% EDTA and could allow infiltration of tetracycline into the dentinal tubule more than 1 mm. NB water did not alter the dentin microhardness compared with 17% EDTA (P .05). At 50-μm depth, CLSM analysis showed no statistically significant difference between 1.5% NaOCl in NB water and 5.25% NaOCl with or without irrigation activation at the coronal, middle, and apical root segments (P .05), ie, these groups had stronger bacterial killing than 1.5% NaOCl (P .05). At deeper levels (100 and 150 μm), higher concentrations of NaOCl were more effective than 1.5% NaOCl with or without NB water. No statistically significant difference was noted between 5.25% NaOCl with and without irrigation activation at most depth levels (P .05).NB water can allow smear layer removal and enhance tubular penetration of medicaments without changing dentin microhardness. In large canal models, NB water appears to improve the tubular disinfection capacity of lower concentration of NaOCl up to 50 μm. On the other hand, the use of irrigation activation (US or XP) did not provide any added disinfection into the dentinal tubules compared with SNI. These results suggest that NB water may be a promising adjunct to endodontic irrigants and medicaments.

Dental stem cells (DSCs) have been shown to possess great potential for multiple biomedical applications, especially for dental tissue regeneration. They are a special type of subpopulation of mesenchymal stem/stromal cells (MSCs) and present subtle differences from other types of MSCs. Therefore, it requires a specialized expertise to isolate, culture, and characterize these cells in vitro and in vivo. The purpose of this chapter is to share our experience in studying these cells. We will describe in detail laboratory protocols outlining how the cells are isolated, cultured, expanded, and characterized using various in vitro cellular and biochemical analyses, as well as an in vivo study model using immunocompromised mice to observe tissue regeneration after transplantation of these DSCs.

Dental stem cells (DSCs) have been shown to possess great potential for multiple biomedical applications, especially for dental tissue regeneration. They are a special type of subpopulation of mesenchymal stem/stromal cells (MSCs) and present subtle differences from other types of MSCs. Therefore, it requires a specialized expertise to isolate, culture, and characterize these cells in vitro and in vivo. The purpose of this chapter is to share our experience in studying these cells. We will describe in detail laboratory protocols outlining how the cells are isolated, cultured, expanded, and characterized using various in vitro cellular and biochemical analyses, as well as an in vivo study model using immunocompromised mice to observe tissue regeneration after transplantation of these DSCs.

Introduction Previously, we have shown that stem cells from apical papilla (SCAPs) can be chemoattracted by stromal cell–derived factor-1α (SDF-1α). The purpose of this study was to investigate the intracellular signaling pathways involved in SDF-1α–mediated migration of SCAPs. Methods Chemotaxis assays were performed to assess the effect of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) signaling pathways in the SDF-1α–mediated migration of SCAPs using inhibitors of PI3K (LY294002) or PKC (GF109203X). The Cell Counting Kit-8 assay (Dojindo Laboratories, Kumamoto, Japan) was used to evaluate the effect of the inhibitors on the proliferation of SCAPs. The expression of focal adhesion–related proteins was examined by immunofluorescence staining and Western blot analysis. Phosphorylation of PI3K subunit p85 and PKC after SDF-1α induction was evaluated by Western blot. Results The inhibition of PI3K or PKC signaling pathways significantly reduced SDF-1α–mediated migration of SCAPs. The inhibitors had no effect on the proliferation of SCAPs. Immunofluorescence analysis revealed that SDF-1α stimulated focal adhesion formation and stress fiber assembly in SCAPs, in addition to up-regulation of the expression of focal adhesion molecules, including p-focal adhesion kinase, p-paxillin, and vinculin. Pretreatment with PI3K or PKC inhibitors before SDF-1α induction significantly inhibited focal adhesion molecule expression. Moreover, increased phosphorylation of p85 and PKC were observed after SDF-1α stimulation, whereas these phosphorylations were down-regulated by the inhibition of PI3K or PKC signaling pathways. Conclusions PI3K and PKC signaling pathways appear to be required for SDF-1α–mediated transmigration of SCAPs. These findings provide insights into the signaling mechanisms that underlie SDF-1α–mediated migration of SCAPs.

Introduction: The aim of this study was to determine the efficiency of 4 irrigation systems in eliminating bacteria in root canals, particularly in dentinal tubules. Methods: Roots of human teeth were prepared to 25/04, autoclaved, and inoculated with Enterococcus faecalis for 3 weeks. Canals were then disinfected by (1) standard needle irrigation, (2) sonically agitating with EndoActivator, (3) XP Endo finisher, or (4) erbium:yttrium aluminum garnet laser (PIPS) (15 roots/group). The bacterial reduction in the canal was determined by MTT assays. For measuring live versus dead bacteria in the dentinal tubules (4 teeth/group), teeth were split open and stained with LIVE/DEAD BackLight. Coronal, middle, and apical thirds of the canal dentin were scanned by using a confocal laser scanning microscope (CLSM) to determine the ratio of dead/total bacteria in the dentinal tubules at various depths. Results: All 4 irrigation protocols significantly eliminated bacteria in the canal, ranging from 89.6% to 98.2% reduction (P < .001). XP Endo had the greatest bacterial reduction compared with other 3 techniques (P < .05). CLSM analysis showed that XP Endo had the highest level of dead bacteria in the Coronal, middle, and apical segments at 50-mu m depth. On the other hand, PIPS had the greatest bacterial killing efficiency at the 150-mu m depth in all 3 root segments. Conclusions: XP Endo appears to be more efficient than other 3 techniques in disinfecting the main canal space and up to 50 Am deep into the dentinal tubules. PIPS appears to be most effective in killing the bacteria deep in the dentinal tubules.

Bone and dentin are mineralized extracellular matrices produced by osteoblasts and odontoblasts, respectively, and their major organic portion is type I collagen. Dentinogenesis Imperfecta (DGI) is one of the most common clinically- and genetically-based disturbances of dentin formation, causing irreversible dentin defects. Among several types of DGI, patients with DGI type II exhibit opalescent dentin with partial or complete pulp obliteration. It has been previously reported that the non-sense mutation (c.133C>T) in Dentin Sialophosphoprotein (DSPP) was identified in DGI type II patients at glutamine residue 45, resulting in the premature stop codon (p.Q45X). DSPP is known to be synthesized as a single gene product and further processed at Gly462-Asp463, resulting in the production of Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP). We hypothesized that the shorter form (Q45X) of N-terminal Dentin Sialoprotein (N-DSP) may cause over-production of type I collagen protein as obliterated pulp is occupied by dentin. To test this hypothesis, we generated mouse recombinant Glutathione-S-Transferase (GST)-N-DSP fusion protein, and the effect of GST-N-DSP was investigated in calvarial bone explant culture and MC3T3-E1 osteoblastic culture systems. Here we show that a significant increase in calvarial bone formation is observed by GST-N-DSP. GST-N-DSP accelerates MC3T3-E1 osteoblast cell growth and proliferation and subsequent osteoblast differentiation by inducing the expression of certain osteogenic markers such as type I collagen, Runx2, Osterix and ATF4. Interestingly, GST-N-DSP significantly enhances dentinogenesis marker gene expression including Dspp and Dmp1 gene expression in non-odontogenic MC3T3-E1 cells. To rule out any artificial effect of GST-tag, we also used the synthetic peptide of N-DSP and confirmed the results of N-DSP peptide were essentially similar to those of GST-N-DSP. Taken together, our data suggest that N-DSP promotes bone formation by accelerating osteoblast cell proliferation and subsequent osteoblast differentiation accompanied by marked up-regulation of the dentin matrix markers, such as Dspp and Dmp1 genes., Highlights • Recombinant N-terminal DSP (N-DSP) protein was generated. • N-DSP mimics the non-sense mutation form of Dentinogenesis Imperfecta type II. • N-DSP enhances bone formation in clavarial ex vivo cultures. • N-DSP accelerates osteoblast proliferation. • N-DSP upregulates type I collagen and Dspp expression in non-odontogenic osteoblasts.

The potency and quality of adult stem cells in our body is reversely proportional to our age. When stem cells were discovered in deciduous teeth, it has drawn much attention to the dental and medical fields. Because these cells are normally from children shedding teeth around ages 6-12, they are relatively more immature stem cells.

There have been significant advancements in regenerative dentistry demonstrated in large animal studies, particularly in the areas of endodontics and periodontics. By using dental stem cells, pulp tissues can be regenerated in empty root canal space, and periodontal pockets can be regenerated to its normal state. To further test its clinical applications and perhaps to provide such patient care service if proven safe and successful, a facility called good manufacturing practice where stem cells are processed is needed. However, such facilities are rare.

The introduction and widespread adoption of induced pluripotent stem cell (iPSC) technology has opened new avenues for craniofacial regenerative medicine. Neural crest cells (NCCs) are the precursor population to many craniofacial structures, including dental and periodontal structures, and iPSC-derived NCCs may, in the near future, offer an unlimited supply of patient-specific cells for craniofacial repair interventions. Here, we used an established protocol involving simultaneous Wnt signaling activation and TGF-β signaling inhibition to differentiate three human iPSC lines to cranial NCCs. We then derived a mesenchymal progenitor cell (NCC-MPCs) population with chondrogenic and osteogenic potential from cranial NCCs and investigated their similarity to widely studied human postnatal dental or periodontal stem/progenitor cells. NCC-MPCs were quite distinct from both their precursor cells (NCCs) and bone-marrow mesenchymal stromal cells, a stromal population of mesodermal origin. Despite their similarity with dental stem/progenitor cells, NCC-MPCs were clearly differentiated by a core set of 43 genes, including ACKR3 (CXCR7), whose expression (both at transcript and protein level) appear to be specific to NCC-MPCs. Altogether, our data demonstrate the feasibility of craniofacial mesenchymal progenitor derivation from human iPSCs through a neural crest-intermediate and set the foundation for future studies regarding their full differentiation repertoire and their in vivo existence.

Introduction Dental pulp stem cells (DPSCs) are multipotent progenitors for biotechnological practices, but the influences of existing restorations on their viability and differentiation are not well-known. This study was aimed to investigate in vivo and in vitro responses of DPSCs to restorative materials. Methods Class I cavities were prepared on molars scheduled to be extracted and then restored with a resin-based composite (RBC), a glass ionomer cement, or zinc oxide eugenol. Intact teeth were used as controls. Twelve molars in each group were extracted on day 7 or day 30 after restorations to assess the early or intermediate pulp responses and were then cut in half. One half was processed for histopathological analysis, and the other was used to isolate DPSCs for a colony-forming unit assay and real-time polymerase chain reaction for NANOG, OCT4, and CD44 expression. Results All restored teeth showed pulp damage at various levels, whereas mild to moderate inflammation persisted in the RBC group until day 30. The existence of DPSCs in the pulp cores of all groups was revealed based on CD44 immunoreactivity. Glass ionomer cement and zinc oxide eugenol did not affect the relative percentages of DPSCs in either early or intermediate stages, whereas RBCs reduced the percentage. The colony-forming units in all restoration groups were comparable with those in the control. Nevertheless, the restorations significantly enhanced OCT4 expression, especially in RBC/day 30. Conclusions Dental restorations cause mild pulp damage but do not affect DPSC viability. RBC decreases DPSC densities but might increase the stemness of surviving DPSCs through an inflammation-stimulation process.

The objective of this study was to determine whether cells from human pulp can be transduced to express the antimicrobial peptide--human β-defensin-2 (HBD-2). Primary human pulp cells and gingival fibroblasts from normal tissue, as well as two mouse cell lines (NIH 3T3 and AT-84) and a human cell line SCC-9 were transduced with a retroviral vector carrying HBD-2 cDNA. ELISA and Northern blot analyses were performed to detect HBD-2 expression by these transduced cells. Antimicrobail assays using recombinant HBD-2 were performed on two caries-associated bacteria Streptococcus mutnas and Lactobacillus acidophilus. The results showed that transduced pulp cells secreted 62.4 ± 27.2 ng/3 days of HBD-2, which was comparable to that by NIH 3T3 (78.0 ± 14.1 ng/4 days), and higher than those by gingival fibroblasts (17.9 ± 7.9 ng/3 days), AT-84 (2.6 ± 1.0 ng/3 days), and SCC-9 (47.6 ± 9.9 ng/3 days). Northern blot analysis showed that the levels of HBD-2 mRNA expression correlated with their protein secretion levels. There was approximately 50% reduction of growth when S. mutans and L. acidophilus were exposed to HBD-2 at 1 µM. Pulp cells appear to be suitable for HBD-2 transduction using retroviral vectors, suggesting a potential for use in controlling pulpal infections.

The goal of this study was to establish mini-swine as a large animal model for stem cell-based pulp regeneration studies. Swine dental pulp stem cells (sDPSCs) were isolated from mini-swine and characterized in vitro. For in vivo studies, we first employed both ectopic and semi-orthotopic study models using severe combined immunodeficiency mice. One is hydroxyapatite-tricalcium phosphate (HA/TCP) model for pulp-dentin complex formation, and the other is tooth fragment model for complete pulp regeneration with new dentin depositing along the canal walls. We found that sDPSCs are similar to their human counterparts exhibiting mesenchymal stem cell characteristics with ability to form colony forming unit-fibroblastic and odontogenic differentiation potential. sDPSCs formed pulp-dentin complex in the HA/TCP model and showed pulp regeneration capacity in the tooth fragment model. We then tested orthotopic pulp regeneration on mini-swine including the use of multi-rooted teeth. Using autologous sDPSCs carried by hydrogel and transplanted into the mini-swine root canal space, we observed regeneration of vascularized pulp-like tissue with a layer of newly deposited dentin-like (rD) tissue or osteodentin along the canal walls. In some cases, dentin bridge-like structure was observed. Immunohistochemical analysis detected the expression of nestin, dentin sialophosphoprotein, dentin matrix protein 1, and bone sialoprotein in odontoblast-like cells lining against the produced rD. We also tested the use of allogeneic sDPSCs for the same procedures. Similar findings were observed in allogeneic transplantation. This study is the first to show an establishment of mini-swine as a suitable large animal model utilizing multi-rooted teeth for further cell-based pulp regeneration studies.

Regenerative endodontic therapy (RET) is currently used to treat immature teeth with necrotic pulp and/or apical periodontitis. However, recently RET has been used to treat mature teeth with necrotic pulp and/or apical periodontitis and resulted in regression of clinical signs and/or symptoms and resolution of apical periodontitis. The purpose of this case report was to describe the potential of using RET to treat 2 mature teeth with persistent apical periodontitis after root canal therapy using RET. Two male patients, one 26-year old and another 12-year old, presented for retreatment of persistent apical periodontitis after root canal treatment of 2 mature teeth (#9 and #19). The gutta-percha fillings in the canals of teeth #9 and #19 were removed with Carvene gutta-percha solvent (Prevest DenPro, Jammu, India) and ProTaper Universal rotary retreatment files (Dentsply Maillefer, Ballaigues, Switzerland). The canals of both teeth were further chemomechanically debrided with rotary retreatment files and copious amounts of sodium hypochlorite irrigation and dressed with Metapaste (Meta Biomed, Chungbuk, Korea). RET was performed on teeth #9 and #19. Periapical bleeding was provoked into the disinfected root canals. The blood clots were covered with mineral trioxide aggregate plugs, and the access cavities were restored with intermediate restorative material. Teeth #9 and #19 showed regression of clinical signs and/or symptoms and healing of apical periodontitis after 13-month and 14-month follow-ups, respectively. Tooth #9 revealed narrowing of the canal space and apical closure by deposition of hard tissue. RET has the potential to be used to retreat teeth with persistent apical periodontitis after root canal therapy.

Introduction Stem cells from the apical papilla (SCAPs) at the apex may be attracted into the root canal space as a cell source for pulp-dentin regeneration. To test this possibility, we used in vitro transmigration models to investigate whether SCAPs can be chemoattracted by the delivery of the chemotactic cytokine stromal cell–derived factor-1α (SDF-1α). Methods We first examined the expression of CXC chemokine receptor 4 (CXCR4) for SDF-1α in the apical papilla and in cultured SCAPs using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and flow cytometric analyses. A standard Transwell migration assay and a 3-dimensional cell migration assay were used to analyze transmigration of SCAPs via the SDF-1α/CXCR4 axis. Results CXCR4 was expressed in the paravascular region of the apical papilla and detected in SCAP cultures. Most cultured SCAPs harbored intracellular CXCR4 (58%–99%, n = 4), whereas only a few cells had detectable CXCR4 on the cell surface (0.3%–2.34%, n = 4). Although SDF-1α had no significant effect on SCAP proliferation, it significantly promoted a higher number of migrated cells; this effect was abolished by anti-CXCR4 antibodies. Interestingly, cell surface CXCR4 on SCAPs was not detectable until after transmigration. The 3-dimensional migration assay revealed that SDF-1α significantly enhanced SCAP migration in the collagen gel. Conclusions SCAPs can be chemoattracted via the SDF-1α/CXCR4 axis, suggesting that SDF-1α may be used clinically to induce CXCR4-expressing SCAPs in the apical papilla to transmigrate into the root canal space as an endogenous cell source for pulp regeneration.

The discovery of dental stem cells provided a new avenue for cell-based tissue engineering and regeneration of dental tissues. These dental tissue–derived mesenchymal stem/stromal cells (MSCs) share characteristics similar to other MSCs, yet, they are subtly different. They are relatively robust stem/progenitor cells, and these cells can proliferate many times in cultures, reaching a large number from a small population at the time of isolation. Since their discovery, tremendous progress has been made demonstrating their potential in dental tissue regeneration in small and large animal models. This chapter will review the recent studies on the characteristics of several dental stem cells and their potential tissue regeneration potential including pulp dentin, periodontal tissue, and whole tooth regeneration.

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Introduction Iatrogenic furcation perforation may occur during the access preparation of the endodontic treatment. This may lead to periodontal defects and subsequent tooth loss. In this case report, we presented a new approach that may help salvage cases with a longstanding furcation involvement and substantial bone loss resulting from perforation. Methods A mandibular molar case that had a furcation perforation and longstanding furcation bone loss with a probing depth of 10 mm in the buccal furcation area. We applied a novel approach, which used both nonsurgical and surgical interventions. We first reaccessed the tooth to reseal the perforation site with MTA followed by a newly designed surgical approach including the use of a stent, a reverse submarginal flap, Emdogain (Straumann USA, Andover, MA), guided bone regeneration, and postoperative isolation of the surgical site. The post-treatment follow-ups with up to 19-month recall showed favorable results with significant bone regeneration at the furcation and the probing depth reduced to 4–5 mm. Conclusions Longstanding furcation perforations with periodontal involvement may be savable and have a better prognosis. This may require a modified flap design to access the defect, guided bone and periodontal regeneration, and postoperative isolation of the surgical defect.

Objective: The purpose of this study was to determine the effectiveness of laser-activated irrigation by photon-induced photoacoustic streaming (PIPS) using Er:YAG laser energy in decontaminating heavily colonized root canal systems in vitro. Materials and methods: Extracted single-rooted human teeth (n=60) were mechanically and chemically prepared, sterilized, inoculated with Enterococcus faecalis for 3 weeks, and randomly assigned to four groups (n=15): Group I (control, no decontamination), Group II (PIPS+6% NaOCl), Group III (PIPS+saline), and Group IV (6% NaOCl). PIPS settings were all preset to 50 μsec pulse, 20 mJ, 15 Hz, for an average power of 0.3 W. After decontamination, the remaining live microbes from all specimens were collected and recovered via plate counting of the colony-forming units (CFUs). Randomized root canal surfaces were examined with scanning electron microscopy and confocal laser microscopy. Mean variance and Dunnett's t test (post-hoc test) comparisons were used to compare mean scores for the three groups with the control group. Results: The CFU analysis showed the following measurements (mean±SE): Group I (control), 336.8±1.8; Group II (PIPS+NaOCl), 0.27±0.21; Group III (PIPS+saline), 225.0±21; and Group IV (NaOCl), 46.9±20.29. Group II had significantly lower CFUs than any other groups (p

The concept of regenerative endodontics has gained much attention in clinical endodontics in the past decade. One aspect of this discipline is the application of revitalization/revascularization therapies for infected and/or necrotic immature pulps in permanent teeth. Following the publication of a case report (Iwaya et al. ), investigators have been rigorously examining the types of tissues formed in the canals as well as exploring strategies to regenerate the pulp-dentine complex in revitalized teeth. This review will provide an update on the types of tissues generated in the canals after revitalization/revascularization therapy in both animal and human studies. The understanding of the role of stem cells and microenvironment in the process of wound healing resulting in either regeneration or repair will be thoroughly discussed. Stem cells and microenvironmental cues introduced into the canal during revitalization/revascularization procedures will be examined. In addition, requirement of a sterile microenvironment in the canal and vital tissue generation in revitalization/revascularization therapy will be emphasized. The challenges that we face and the hopes that we have in revitalization/revascularization therapy for regenerative endodontics will be presented.

Zhu W, Zhu X, Huang GT-J, Cheung GSP, Dissanayaka WL, Zhang C. Regeneration of dental pulp tissue in immature teeth with apical periodontitis using platelet-rich plasma and dental pulp cells. International Endodontic Journal, 46, 962–970, 2013. Aim To investigate the new tissues growing into the pulp space of immature dog teeth that were infected, disinfected and filled with blood clot (BC), dental pulp cells (DPCs), platelet-rich plasma (PRP) or a combination of DPCs and PRP in immature dog teeth with apical periodontitis. Methodology Fifty-six immature roots from mandibular premolars of four beagles were divided into four experimental groups (n = 40) and two control groups. After the induction of apical periodontitis, the root canals of experimental groups were disinfected with NaOCl irrigation and a tri-antibiotic paste medication. The canals were then filled with different materials according to the experimental group: BC group, DPCs group, PRP group or DPCs + PRP group. Access cavities were sealed with MTA and composite. Radiographs were taken after 90 days, and the jaws including the teeth were processed for histologic analysis. The data were statistically analysed using chi-square evaluation and Student’s t-test. Results Radiographic analyses demonstrated no significant difference between experimental groups in periradicular bone healing (P > 0.05), whilst those groups that used DPCs produced a significantly greater root thickening (P < 0.01). The histologic evaluation showed that the groups with PRP formed more tissues in the canals (P = 0.01). The groups with DPCs had substantially more mineralized tissue formation in the canal than those without DPCs, especially in the apical third. In DPCs + PRP group, bone-like tissue grew into the canal space from the periapical tissue. Conclusions A combination of DPCs + PRP increased vital tissue regeneration within the root canals of immature teeth associated with apical periodontitis.

There are two types of approaches to regenerate tissues: cell-based and cell-free. The former approach is to introduce exogenous cells into the host to regenerate tissues, and the latter is to use materials other than cells in an attempt to regenerate tissues. There has been a significant advancement in stem cell-based pulp and dentin regeneration research in the past few years. Studies in small and large animals have demonstrated that pulp/dentin-like tissues can be regenerated partially or completely in the root canal space with apical openings of 0.7-3.0 mm using dental pulp stem cells, including stem cells from apical papilla (SCAP) and subpopulations of pulp stem cells. Bone marrow mesenchymal stem cells (BMMSCs) and adipose tissue-derived MSCs (ADMSCs) have also been shown to regenerate pulp-like tissue. In contrast, the cell-free approach has not produced convincing evidence on pulp regeneration. However, one crucial concept has not been considered nor defined in the field of pulp/dentin regeneration and that is the critical size defect of dentin and pulp. Without such consideration and definition, it is difficult to predict or anticipate the extent of cell-free pulp regeneration that would occur. By reasoning, cell-free therapy is unlikely to regenerate an organ/tissue after total loss. Similarly, after a total loss of pulp, it is unlikely to regenerate without using exogenously introduced cells. A cell homing approach may provide a limited amount of tissue regeneration. Although stem cell-based pulp/dentin regeneration has shown great promise, clinical trials are difficult to launch at present. This article will address several issues that challenge and hinder the clinical applications of pulp/dentin regeneration which need to be overcome before stem cell-based pulp/dentin regeneration can occur in the clinic.

Introduction: The purpose of this study was to investigate whether combined and concerted delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) enhances odonto/osteogenic differentiation of human dental pulp stem cells (DPSCs) in vitro. Methods: Various concentrations of VEGF and/or BMP-2 with or without the presence of odonto/osteogenic medium (OM) were added into DPSC cultures for 21 days. The mineral formation in cultures was evaluated using alizarin red stain (ARS). Optimal concentrations of VEGF and BMP-2 were codelivered to DPSCs for total of 21 days with the following experimental groups: (1) group 1: OM only, (2) group 2: OM + VEGF, (3) group 3: OM + BMP-2, and (4) group 4: OM + VEGF + BMP-2 (Subgroup 4a: VEGF present the first 7 days, 4b: BMP-2 present the last 14 days, and 4c, both present for 21 days). Cultures were then subjected to quantitative ARS analysis or harvested for quantitative polymerase chain reaction analysis for the expression of core-binding factor alpha 1 (CBFA1), alkaline phosphatase (ALP), and dentin matrix protein 1 (DMP-1). Results: No mineral formation was detected by ARS when VEGF and/or BMP-2 were used without OM. OM + VEGF, but not OM + BMP-2, formed more mineralization than OM (P < .05). In the codelivery groups, the highest mineralization was observed in OM + VEGF and subgroup 4a compared with OM or the other groups (P < .05). Quantitative polymerase chain reaction analysis showed that CBFA1, ALP, and DMP-1 levels were higher in groups 2, 3, and 4a compared with 4b and 4c (P < .05). CBFA1 expressed higher in groups 2, 3, and 4a compared with OM (P < .05). For ALP expression, only subgroup 4a expressed higher than OM (P < .05). No difference was detected between groups 2 and 3 (P >.05) in the expression of the 3 genes. Conclusions: VEGF addition in the early phase rather than a continuous presence of both VEGF and BMP-2 enhances odonto/osteogenic differentiation of DPSCs.

Introduction: The aim of this study was to quantify vascular network formation capacity after angiogenic induction of human and swine dental pulp stem cells (DPSCs) in comparison with endothelial cells. Methods: Primary human DPSCs or swine DPSCs were induced in endothelial growth medium for 7 days. The expression of the endothelial marker von Willebrand factor was determined by immunostaining. Induced DPSCs (iDPSCs) and noninduced DPSCs (niDPSCs) were seeded at different cell numbers onto Matrigel (BD Biosciences, San Jose, CA) for vascular network formation assays and analyzed after 4, 8, 12, and 18 hours in comparison with human microvascular endothelial cells (hMECs). Quantitative analysis of vascular tubule formation was performed using Image) software (National Institutes of Health, Bethesda, MD). The vascular network formation was also conducted by coculturing of niDPSCs and iDPSCs. Results: Von Willebrand factor was detected by immunofluorescence in both niDPSCs and iDPSCs (human and swine). Time-lapse microscopic observation showed that the vascular network was formed by iDPSCs but not niDPSCs. After 4 hours, iDPSCs showed vascular network formation, whereas niDPSCs started to aggregate and formed clusters. Human iDPSCs displayed a similar capacity to form vascular networks in Matrigel compared with hMECs based on quantitative analysis. Swine iDPSCs had a higher capacity compared with human iDPSCs or hMECs (P < .05) in forming the network structures including segments, nodes, and mesh. A coculture experiment showed that human niDPSCs colocalized on the angiogenic tubules and vascular networks that were formed by human iDPSCs. Conclusions: Our findings indicate that iDPSCs in combination with their non induced counterparts may be used as a future clinical strategy for enhancing angiogenesis during the process of pulp-dentin regeneration.

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1−/CD146+ and STRO-1−/CD146− subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were down-regulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

We previously reported the presence of mesenchymal stem/progenitor cells (MSCs) in inflamed pulp tissue. Here we asked whether MSCs also exist in inflamed periapical tissues resulting from endodontic infection. The objectives of this study were to detect the expression of MSC markers in periapical inflammatory tissues and to characterize isolated cells from these tissues.Human periapical inflammatory tissues were collected and processed to detect MSC marker expression by immunohistochemistry. Cells were isolated and tested for cell surface marker expression by using flow cytometry and examined for multiple differentiation potential into osteogenic and adipogenic pathways. In vivo formation of mineralized tissues was assessed in a mouse model.Immunohistochemistry showed positive staining for MSC markers STRO-1, CD90, and CD146. Isolated cells at passage 0 appeared as typical fibroblastic cells, and a few cells formed colony-forming unit-fibroblasts (CFU-Fs). After passaging, the CFU-F forming ability diminished dramatically, and the population doubling was up to 26. Flow cytometry data showed that these cells at passage 2 expressed low levels of STRO-1 and CD146 and moderate to high levels of CD90, CD73, and CD105. At passage 6, the levels of these markers decreased. When incubated in specific differentiation medium, cells demonstrated a strong osteogenic but weak adipogenic capacity. After in vivo cell transplantation, mineralized tissues formed in immunocompromised mice.Human periapical inflammatory tissues expressed MSC markers, suggesting the presence of MSCs. Isolated cells exhibited typical mesenchymal cell immunophenotype with a capacity to form mineralized matrix in vitro and in vivo.

Generation of induced pluripotent stem (iPS) cells using defined factors has been considered a ground-breaking step towards establishing patient-specific pluripotent stem cells for various applications. The isolation of human embryonic stem (ES) cells set the standard that pluripotent stem cells are attainable as potentially immortal cells for regeneration of many types of tissues. Different approaches have been tested to obtain pluripotent stem cells by circumventing the need for embryos. iPS cells appear to be an ideal substitute for ES cells. Since the first demonstration of creating iPS cells in 2006, tremendous efforts have been made into improving iPS cell generation methods and understanding the reprogramming mechanism as well as the nature of iPS cells. To improve iPS cell generation, several approaches have been taken: (1) eliminate the viral vector integration after delivering the defined factors; (2) select different cell types that more effectively give rise to iPS cells; (3) use of chemicals to facilitate reprogramming; (4) use of protein factors to reprogram cells. The iPS cells are also being rigorously characterized in comparison to ES cells. All these efforts are made for the purpose of making iPS cells closer to clinical applications. This article will give an overview of the following areas: (1) mechanisms of iPS cell derivation; (2) characterization of iPS cells; (3) iPS cells for cell-based therapy; and (4) iPS cells for studying disease mechanism. Questions as to what aspects of iPS cells require further understanding before they may be put to clinical use are also discussed.

Background: Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims: To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials & methods: DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results: The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. Conclusion: DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo.