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Pulp regeneration after transplantation of mobilized dental pulp stem cells (MDPSCs) declines in the aged dogs due in part to the chronic inflammation and/or cellular senescence. Eotaxin-1/C-C motif chemokine 11 (CCL11) is an inflammation marker via chemokine receptor 3 (CCR3). Moreover, CCR3 antagonist (CCR3A) can inhibit CCL11 binding to CCR3 and prevent CCL11/CCR3 signaling. The study aimed to examine the effect of CCR3A on cellular senescence and anti-inflammation/immunomodulation in human periodontal ligament cells (HPDLCs). The rejuvenating effects of CCR3A on neurite extension and migratory activity to promote pulp regeneration in aged dog teeth were also evaluated. In vivo, the amount of regenerated pulp tissues was significantly increased by transplantation of MDPSCs with CCR3A compared to control without CCR3A. In vitro, senescence of HPDLCs was induced after p-Cresol exposure, as indicated by increased cell size, decreased proliferation and increased senescence markers, p21 and IL-1β. Treatment of HPDLCs with CCR3A prevented the senescence effect of p-Cresol. Furthermore, CCR3A significantly decreased expression of CCL11, increased expression of immunomodulatory factor, IDO, and enhanced neurite extension and migratory activity. In conclusion, CCR3A protects against p-Cresol-induced cellular senescence and enhances rejuvenating effects, suggesting its potential utility to stimulate pulp regeneration in the aged teeth.

Regenerative cell therapy using autologous dental pulp stem cells (DPSCs) in mature single-rooted teeth is a potential alternative to traditional endodontic treatment. However, there is no evidence supporting the use of DPSCs in multirooted teeth. This case report aimed to demonstrate the feasibility and outcomes of pulp regenerative cell therapy in mature multirooted molars, which typically have a higher prevalence of apical deltas. A 26-year-old male and a 29-year-old male were referred for the pulp regeneration of their maxillary molars. After access preparation and establishing apical patency, root canal preparation and disinfection were performed. Autologous DPSCs were isolated from extracted third molars, cultured according to the guidelines of good manufacturing practice, and transplanted into the prepared root canals with granulocyte colony-stimulating factor in atelocollagen. The access cavity was sealed with Biodentine and composite resin. Clinical evaluation during the follow-up period of 48 weeks and laboratory evaluation after 4 weeks revealed no adverse events or evidence of systemic toxicity. After 48 weeks, radiographs and cone-beam computed tomography showed no periapical radiolucency. The teeth showed a positive response to electric pulp testing in 4 weeks in both cases. The signal intensities on magnetic resonance imaging of the regenerated pulp tissue in the affected teeth were comparable to those of the normal pulp in adjacent teeth after 24 weeks. This report of 2 cases demonstrates the utility of DPSCs and the feasibility of pulp regenerative cell therapy in multirooted molars.

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.

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.

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. Our clinical study has demonstrated safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. It is well known that the oxygen concentration is closely linked to the maintenance of stemness. Thus, in this investigation, hypoxia-preconditioned DPSCs (hpDPSCs) was characterized to develop and improve the clinical utility for regeneration of dental pulp in endodontics.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 were 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 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 up-regulated 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 hpDPSCs improved stem cell properties compared to MDPSCs, suggesting their potential clinical utility for pulp regeneration.

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.

The effects of transplanted human dental pulp-derived cells (DPCs) on peripheral nerve regeneration were studied in a rat model of sciatic nerve crush injury. In one group, DPCs were transplanted into the compression site (cell transplantation group); the control group underwent no transplantation (crushed group). Sciatic nerve regeneration was determined based on the recovery of motor function and histological and immunohistochemical analyses. The cell transplantation group showed improved motor function compared with the crushed group using the CatWalk XT system, which corresponded to a higher ratio of tibialis to anterior muscle weight 14 days after surgery. Histological analysis revealed a smaller interspace area and few vacuoles in the sciatic nerve after cell transplantation compared with the crushed group. The myelin sheath was visualized with Luxol Fast Blue (LFB) staining and anti-myelin basic protein (anti-MBP) antibody labeling; the percentages of LFB- and MBP-positive areas were higher in the cell transplantation group than in the crushed group. Human mitochondria-positive cells were also identified in the sciatic nerve at the transplantation site 14 days after surgery. Taken together, the observed correlation between morphological findings and functional outcomes following DPC transplantation indicates that DPCs promote peripheral nerve regeneration in rats.

Pulp regeneration by stem cell transplantation declines due to age-related reduction. We hypothesized that administration of a cytokine together with the cell transplantation may improve the stem cell niche microenvironment and promote regeneration. CCL11 is implicated as a factor in aging. This investigation was performed to investigate the changes in the quality of the regenerated pulp by administration of CCL11 antibody in the aged mice and elucidate the underlying mechanisms.Mobilized dental pulp stem cell (MDPSC) transplants were characterized in an ectopic tooth root transplantation model in both the aged and young mice. The amount of regenerated pulp tissue was analyzed in the transplants with continuous administration of CCL11 antibody compared with those without the antibody administration. Blood CCL11 levels were assessed at the onset of the experiment. Furthermore, immunostaining of CD68 together with CD11c or CD206 for M1 and M2 macrophage, respectively, were performed. Each double-positive cell count of M1 and M2 macrophages and M1/M2 ratio in the transplants with administration were compared with those without administration both in the aged and young mice.The administration of CCL11 antibody enhanced pulp regeneration and significantly reduced the blood CCL11 level in the aged mice. As the number of M1 macrophages decreased, the M1/M2 ratio in the treated aged mouse was less than that in the untreated aged mouse. There was, however, significant difference between the treated aged mouse and the untreated young mouse.CCL11 antibody has the potential to enhance and stimulate pulp regeneration in the aged mice.

The juxta-oral organ is a persistent bilateral organ in the mammalian bucca. It consists of epithelial parenchyma and surrounding mesenchymal sheath with connective tissue rich in nerve fibers and sensory receptors between them. The organ develops from the embryonic oral epithelium and is separated from the oral mucosa. The morphology of this organ is very different in mice compared to humans. Although several reports have described the histology of the juxta-oral organ in rodents, its developmental changes have rarely been reported. In the present study, histological development and ultrastructure of the juxta-oral organ, with special attention to its epithelial parenchyma and mesenchymal sheath, were investigated in mice during several developmental stages. Immunohistochemical staining for pan-cytokeratin (CK) on serial paraffin sections of the head allowed easier detection of this organ. The juxta-oral organ was innervated by the buccal nerve from an early stage of organogenesis at E13.5. The organ was located adjacent to the fascia of the masticatory muscles during all developmental stages examined. The parenchymal cells were immunohistochemically positive for p63 and CK14 in both newborn and adult mice, suggesting that these epithelial cells possess characteristics of the basal cells of the stratified epithelium. Furthermore, the parenchyma was ensheathed by layers of mesenchymal cells with perineurial characteristics as shown by immunohistochemical staining and electron microscopy. Unique sensory endings and bundles of elastic fibers were observed between the epithelial parenchyma and mesenchymal sheath. The present findings concerning the structure of mouse juxta-oral organ, especially that of epithelial parenchyma and mesenchymal sheath provide baseline knowledge for the further understanding of the unique morphological features, developmental changes and functional details of this organ.

Magnetic resonance imaging (MRI) has recently been used for the evaluation of dental pulp anatomy, vitality, and regeneration. This study reviewed the recent use of MRI in the endodontic field. Literature published from January 2000 to March 2017 was searched in PubMed using the following Medical Subject Heading (MeSH) terms: (1) MRI and (dental pulp anatomy or endodontic pulp); (2) MRI and dental pulp regeneration. Studies were narrowed down based on specific inclusion criteria and categorized as in vitro, in vivo, or dental pulp regeneration studies. The MRI sequences and imaging findings were summarized. In the in vitro studies on dental pulp anatomy, T1-weighted imaging with high resolution was frequently used to evaluate dental pulp morphology, demineralization depth, and tooth abnormalities. Other sequences such as apparent diffusion coefficient mapping and sweep imaging with Fourier transformation were used to evaluate pulpal fluid and decayed teeth, and short-T2 tissues (dentin and enamel), respectively. In the in vivo studies, pulp vitality and reperfusion were visible with fat-saturated T2-weighted imaging or contrast-enhanced T1-weighted imaging. In both the in vitro and in vivo studies, MRI could reveal pulp regeneration after stem cell therapy. Stem cells labeled with superparamagnetic iron oxide particles were also visible on MRI. Angiogenesis induced by stem cells could be confirmed on enhanced T1-weighted imaging. MRI can be successfully used to visualize pulp morphology as well as pulp vitality and regeneration. The use of MRI in the endodontic field is likely to increase in the future.

Background Experiments have previously demonstrated the therapeutic potential of mobilized dental pulp stem cells (MDPSCs) for complete pulp regeneration. The aim of the present pilot clinical study is to assess the safety, potential efficacy, and feasibility of autologous transplantation of MDPSCs in pulpectomized teeth. Methods Five patients with irreversible pulpitis were enrolled and monitored for up to 24 weeks following MDPSC transplantation. The MDPSCs were isolated from discarded teeth and expanded based on good manufacturing practice (GMP). The quality of the MDPSCs at passages 9 or 10 was ascertained by karyotype analyses. The MDPSCs were transplanted with granulocyte colony-stimulating factor (G-CSF) in atelocollagen into pulpectomized teeth. Results The clinical and laboratory evaluations demonstrated no adverse events or toxicity. The electric pulp test (EPT) of the pulp at 4 weeks demonstrated a robust positive response. The signal intensity of magnetic resonance imaging (MRI) of the regenerated tissue in the root canal after 24 weeks was similar to that of normal dental pulp in the untreated control. Finally, cone beam computed tomography demonstrated functional dentin formation in three of the five patients. Conclusions Human MDPSCs are safe and efficacious for complete pulp regeneration in humans in this pilot clinical study.

Stemming from in vitro and in vivo pre-clinical and human models, tissue-engineering-based strategies continue to demonstrate great potential for the regeneration of the pulp-dentin complex, particularly in necrotic, immature permanent teeth. Nanofibrous scaffolds, which closely resemble the native extracellular matrix, have been successfully synthesized by various techniques, including but not limited to electrospinning. A common goal in scaffold synthesis has been the notion of promoting cell guidance through the careful design and use of a collection of biochemical and physical cues capable of governing and stimulating specific events at the cellular and tissue levels. The latest advances in processing technologies allow for the fabrication of scaffolds where selected bioactive molecules can be delivered locally, thus increasing the possibilities for clinical success. Though electrospun scaffolds have not yet been tested in vivo in either human or animal pulpless models in immature permanent teeth, recent studies have highlighted their regenerative potential both from an in vitro and in vivo ( i.e., subcutaneous model) standpoint. Possible applications for these bioactive scaffolds continue to evolve, with significant prospects related to the regeneration of both dentin and pulp tissue and, more recently, to root canal disinfection. Nonetheless, no single implantable scaffold can consistently guide the coordinated growth and development of the multiple tissue types involved in the functional regeneration of the pulp-dentin complex. The purpose of this review is to provide a comprehensive perspective on the latest discoveries related to the use of scaffolds and/or stem cells in regenerative endodontics. The authors focused this review on bioactive nanofibrous scaffolds, injectable scaffolds and stem cells, and pre-clinical findings using stem-cell-based strategies. These topics are discussed in detail in an attempt to provide future direction and to shed light on their potential translation to clinical settings.

Introduction Matrix metalloproteinase (MMP)-3 is a member of the MMP family that degrades the extracellular matrix. Application of MMP-3 to injured pulp tissue induces angiogenesis and wound healing, but its anti-inflammatory effects are still unclear. Here, we evaluated the anti-inflammatory functions of MMP-3 in vitro and in vivo. Methods Nitric oxide and inflammatory mediator synthesis in macrophages activated by lipopolysaccharide (LPS) was measured in the presence or absence of MMP-3. The mouse Mmp3 (mMmp3) expression vector containing full length cDNA sequence of mMmp3 or cDNA sequence of mMmp3 missing the signal peptide and pro-peptide regions was transfected to RAW264, a mouse macrophage cell line, and NO synthesis and inflammatory mediator expression were evaluated. Pulpal inflammation was histologically and immunohistochemically evaluated in a rat model of incisor pulpitis induced by the application of LPS for 9 hours in the presence or absence of MMP-3. Results NO and pro-inflammatory mediator synthesis promoted by LPS was significantly down-regulated by MMP-3 in vitro. The full length of mMmp3 down-regulated the LPS-induced NO synthesis and chemical mediator mRNA expression, however the mMmp3 missing the signal peptide failed to block the NO synthesis induced by LPS. The numbers of major histocompatibility complex class II+ and CD68+ cells, which infiltrated into the rat incisor pulp tissues in response to the topical application of LPS, were significantly decreased by the application of MMP-3 in vivo. Conclusions These results indicate that MMP-3 possesses anti-inflammatory functions, suggesting its potential utility as an anti-inflammatory agent for pulpal inflammation.

The mechanical induction of cell differentiation is well known. However, the effect of mechanical compression on odontoblastic differentiation remains to be elucidated. Thus, we first determined the optimal conditions for the induction of human dental pulp stem cells (hDPSCs) into odontoblastic differentiation in response to mechanical compression of three-dimensional (3D) scaffolds with dentinal tubule-like pores. The odontoblastic differentiation was evaluated by gene expression and confocal laser microscopy. The optimal conditions, which were: cell density, 4.0 × 105 cells/cm2 ; compression magnitude, 19.6 kPa; and loading time, 9 h, significantly increased expression of the odontoblast-specific markers dentine sialophosphoprotein (DSPP) and enamelysin and enhanced the elongation of cellular processes into the pores of the membrane, a typical morphological feature of odontoblasts. In addition, upregulation of bone morphogenetic protein 7 (BMP7) and wingless-type MMTV integration site family member 10a (Wnt10a) was observed. Moreover, the phosphorylation levels of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 were also enhanced by mechanical compression, indicating the involvement of the MAPK signalling pathway. It is noteworthy that human mesenchymal stem cells (MSCs) derived from bone marrow and amnion also differentiated into odontoblasts in response to the optimal mechanical compression, demonstrating the importance of the physical structure of the scaffold in odontoblastic differentiation. Thus, odontoblastic differentiation of hDPSCs is promoted by optimal mechanical compression through the MAPK signalling pathway and expression of the BMP7 and Wnt10a genes. The 3D biomimetic scaffolds with dentinal tubule-like pores were critical for the odontoblastic differentiation of MSCs induced by mechanical compression. Copyright © 2014 John Wiley & Sons, Ltd.

Stem cell therapy is a potential strategy to regenerate the dentin-pulp complex, enabling the conservation and restoration of functional teeth. We assessed the efficacy and safety of pulp stem cell transplantation as a prelude before the initiation of clinical trials. Granulocyte-colony stimulating factor (G-CSF) induces subsets of dental pulp stem cells to form mobilized dental pulp stem cells (MDPSCs). Good manufacturing practice is a prerequisite for the isolation and expansion of MDPSCs that are enriched in stem cells, expressing a high level of trophic factors with properties of high proliferation, migration, and antiapoptotic effects and endowed with regenerative potential. The quality of clinical-grade MDPSCs was assured by the absence of abnormalities/aberrations in karyotype and the lack of tumor formation after transplantation in immunodeficient mice. Autologous transplantation of MDPSCs with G-CSF in pulpectomized teeth in dogs augmented the regeneration of pulp tissue. The combinatorial trophic effects of MDPSCs and G-CSF on cell migration, antiapoptosis, immunosuppression, and neurite outgrowth were also confirmed in vitro. Furthermore, MDPSCs from the aged donors were as potent as the young donors. It is noteworthy that there were no significant age-related changes in biological properties such as stability, regenerative potential, and expression of the senescence markers in MDPSCs. On the other hand, autologous transplantation of MDPSCs with G-CSF induced less regenerated pulp tissue in the aged dogs compared with the young dogs. In conclusion, the preclinical safety, feasibility, and efficacy of pulp regeneration by MDPSCs and G-CSF were established. Therefore, the standardization and establishment of regulatory guidelines for stem cell therapy in clinical endodontics is now a reality.

Dental pulp-derived stem cells (DPSCs) are considered to be of great promise for use in tissue repair and regenerative medicine. DPSCs can easily be collected from discarded teeth with little ethical concerns and harvested in a minimally invasive and safe manner. However, unfractionated clonogenic DPSCs are heterogenous and have variations in their phenotype. In this review paper, we summarize further isolation methods of DPSC subpopulations including immunoselection methods and a granulocyte colony-stimulating factor (G-CSF) gradient mobilization method for therapeutic clinical applications. The fractionated DPSC subpopulations exhibit stem cell properties in vitro: (i) high expression of pluripotency markers, Oct3/4, Nanog, and Sox2; (ii) high stability in long-term expansion; (iii) multi-lineage differentiation capacity; (iv) high migratory activity; and (v) high expression of trophic factors to enhance proliferation, migration, and anti-apoptotic and immunomodulatory effects as well as angiogenesis and neurite extension. DPSC subpopulations have higher angiogenic, neurogenic, and regenerative potential compared with bone marrow stem cells and adipose stem cells, presenting an alternate versatile stem cell source for cellular therapies. Preclinical efficacy of DPSC subpopulations has also been investigated in various tissue/organ disease models including pulpitis, and currently a few clinical trials are underway to determine their safety and efficacy. Therefore, the major aim of this review is to highlight the recent progress in DPSC biology, trends in preclinical regenerative studies, and future perspectives.

Mesenchymal stem cells (MSCs) have been used for cell therapy in various experimental disease models. However, the regenerative potential of MSCs from different tissue sources and the influence of the tissue niche have not been investigated. In this study, we compared the regenerative potential of dental pulp, bone marrow and adipose tissue-derived CD31(-) side population (SP) cells isolated from an individual porcine source. Pulp CD31(-) SP cells expressed the highest levels of angiogenic/neurotrophic factors and had the highest migration activity. Conditioned medium from pulp CD31(-) SP cells produced potent anti-apoptotic activity and neurite outgrowth, compared to those from bone marrow and adipose CD31(-) SP cells. Transplantation of pulp CD31(-) SP cells in a mouse hindlimb ischemia model produced higher blood flow and capillary density than transplantation of bone marrow and adipose CD31(-) SP cells. Motor function recovery and infarct size reduction were greater with pulp CD31(-) SP cells. Pulp CD31(-) SP cells induced maximal angiogenesis, neurogenesis and pulp regeneration in ectopic transplantation models compared to other tissue sources. These results demonstrate that pulp stem cells have higher angiogenic, neurogenic and regenerative potential and may therefore be superior to bone marrow and adipose stem cells for cell therapy.

Recent studies have demonstrated that culture under hypoxia has beneficial effects on mesenchymal stem cells (MSCs). However, there are limitations to achieving a stable condition in conventional hypoxic CO2 incubators. DPSCs are a unique type of MSCs which are promising in many regenerative therapies. In this study, we investigated the ideal hypoxic culture environment for DPSCs using a new system that can provide controlled O2 environment. The effects of hypoxia (3%, 5%) on the stemness properties of DPSCs. Their morphology, proliferation rate, expression of stem cell markers, migration ability, mRNA expression of angiogenic/neurotrophic factors and immunomodulatory genes were evaluated and compared. Additionally, the effect of the discrete secretome on proliferation, migration, and neurogenic induction was assessed. Hypoxic DPSCs were found to be smaller in size and exhibited larger nuclei. 5% O2 significantly increased the proliferation rate, migration ability, expression of stem cell markers (CXCR4 and G-CSFR), and expression of SOX2, VEGF, NGF, and BDNF genes of DPSCs. Moreover, secretome collected from 5%O2 cultures displayed higher stimulatory effects on proliferation and migration of NIH3T3 cells and on neuronal differentiation of SH-SY5Y cells. These results demonstrate that 5%O2 may be ideal for enhancing DPSCs growth, stem cell properties, and secretome trophic effect.

Background The critical challenge in tissue engineering is to establish an optimal combination of stem cells, signaling morphogenetic molecules, and extracellular matrix scaffold/microenvironment. The extracellular matrix components of teeth may be reconstituted as an inductive microenvironment in an ectopic tooth transplantation bioassay. Thus, the isolation and identification of the chemical components of the inductive microenvironment in pulp/dentin regeneration will accelerate progress towards the goal of tissue engineering of the tooth. Methods The teeth demineralized in 0.6 M hydrochloric acid were sequentially extracted by 4.0 M guanidine hydrochloride (GdnHCl), pH 7.4, and 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 7.4. The extracted teeth were transplanted into an ectopic site in severe combined immunodeficiency (SCID) mice with mobilized dental pulp stem cells (MDPSCs). The unextracted tooth served as a positive control. Furthermore, the soluble components for the inductive microenvironment, the GdnHCl extracts, or the EDTA extracts together with or without MDPSC conditioned medium (CM) were reconstituted systematically with autoclaved teeth in which the chemical components were completely inactivated and only the physical microenvironment was preserved. Their pulp/dentin regenerative potential and angiogenic potential were compared 28 days after ectopic tooth transplantation by histomorphometry and real-time RT-PCR analysis. Results Expression of an odontoblastic marker, enamelysin, and a pulp marker, thyrotropin-releasing hormone degrading enzyme (TRH-DE), was lower, and expression of a periodontal cell marker, anti-asporin/periodontal ligament-associated protein 1 (PLAP-1), was higher in the transplant of the EDTA-extracted teeth compared with the GdnHCl-extracted teeth. The autoclaved teeth reconstituted with the GdnHCl extracts or the EDTA extracts have weak regenerative potential and minimal angiogenic potential, and the CM significantly increased this potential. Combinatorial effects of the EDTA extracts and the CM on pulp/dentin regeneration were demonstrated in vivo, consistent with their in-vitro effects on enhanced proliferation, migration, and odontoblastic differentiation. Conclusions The EDTA-extracted teeth demonstrated significantly lower pulp/dentin regenerative potential compared with the GdnHCl-extracted teeth. The EDTA soluble chemical components when reconstituted with the physical structure of autoclaved teeth serve as an inductive microenvironment for pulp/dentin regeneration, promoting cell proliferation, migration, and odontoblastic differentiation.

Dental pulp stem cells (DPSCs) are reported as sources of mesenchymal stem cells (MSCs). MSCs are used as cell therapy options for various diseases. The present study examined the healing effects of DPSC injection on damaged bladder tissue in a chemically induced cystitis rat model. Cystitis was induced by hydrochloride injection into the bladder of female F344/NSlc rats. On the following day, DPSCs suspended in phosphate-buffered saline (PBS) were injected into the bladder and maintained for 1 h (DPSC injection group), while PBS alone was injected as the standard for comparison (PBS injection group). After 2 days following injection, considerable submucosal edema, vascular structure destruction, hemorrhage, and inflammatory cell invasion were observed both in the DPSC and PBS injection groups, with no difference in their degree of submucosal edema and hemorrhage. Six days after injection, vascular structure regeneration was observed in both groups; however, unlike the DPSC injection group, the PBS injection group showed traces of submucosal edema and hemorrhage. These results correlated with tissue concentrations of myeloperoxidase (MPO) and the inflammatory cytokines IL-1β, IL-6, and TNF-α. Furthermore, the intercontraction interval was prolonged, and the frequency of nociceptive behaviors was reduced in the DPSC injection group compared with the PBS injection group. DPSCs were found on the bladder epithelium until day 3 after injection. In the DPSC-conditioned media (CM), the trophic factors FGF-2, VEGF, and the C-C and C-X-C families of chemokines were detected. The results of DPSC injection into the cystitis rat model suggested that the injected cells promote the healing of the damaged bladder tissue by exerting various trophic effects while localizing on the bladder epithelium and that MSC injection is a potential novel therapy for interstitial cystitis/painful bladder syndrome.

Insights into the understanding of the influence of the age of MSCs on their cellular responses and regenerative potential are critical for stem cell therapy in the clinic. We have isolated dental pulp stem cells (DPSCs) subsets based on their migratory response to granulocyte-colony stimulating factor (G-CSF) (MDPSCs) from young and aged donors. The aged MDPSCs were efficiently enriched in stem cells, expressing high levels of trophic factors with high proliferation, migration and anti-apoptotic effects compared to young MDPSCs. In contrast, significant differences in those properties were detected between aged and young colony-derived DPSCs. Unlike DPSCs, MDPSCs showed a small age-dependent increase in senescence-associated β-galactosidase (SA-β-gal) production and senescence markers including p16, p21, Interleukin (IL)-1β, -6, -8, and Groα in long-term culture. There was no difference between aged and young MDPSCs in telomerase activity. The regenerative potential of aged MDPSCs was similar to that of young MDPSCs in an ischemic hindlimb model and an ectopic tooth root model. These results demonstrated that the stem cell properties and the high regenerative potential of MDPSCs are independent of age, demonstrating an immense utility for clinical applications by autologous cell transplantation in dental pulp regeneration and ischemic diseases.

The secretome obtained from stem cell cultures contains an array of neurotrophic factors and cytokines that might have the potential to treat neurodegenerative conditions. Alzheimer’s disease (AD) is one of the most common human late onset and sporadic neurodegenerative disorders. Here, we investigated the therapeutic potential of secretome derived from dental pulp stem cells (DPSCs) to reduce cytotoxicity and apoptosis caused by amyloid beta (Aβ) peptide. We determined whether DPSCs can secrete the Aβ-degrading enzyme, neprilysin (NEP), and evaluated the effects of NEP expression in vitro by quantitating Aβ-degrading activity. The results showed that DPSC secretome contains higher concentrations of VEGF, Fractalkine, RANTES, MCP-1, and GM-CSF compared to those of bone marrow and adipose stem cells. Moreover, treatment with DPSC secretome significantly decreased the cytotoxicity of Aβpeptide by increasing cell viability compared to nontreated cells. In addition, DPSC secretome stimulated the endogenous survival factor Bcl-2 and decreased the apoptotic regulator Bax. Furthermore, neprilysin enzyme was detected in DPSC secretome and succeeded in degradingAβ1–42in vitro in 12 hours. In conclusion, our study demonstrates that DPSCs may serve as a promising source for secretome-based treatment of Alzheimer’s disease.

Introduction Pulp stem/progenitor cells have been successfully transplanted after pulpectomy in dogs and have led to pulp regeneration. The regenerated pulp tissue was investigated by the qualitative and quantitative protein expression patterns in comparison with those of normal pulp. There is an unmet need for a quality standard for regenerated pulp tissue. Methods Three distinct human CD105 + stem/progenitor cells from dental pulp, bone marrow, and amnion were compared by 2-dimensional electrophoresis and the differential proteomic expression profiles. The protein identified with high confidence in pulp CD105 + cells was examined by immunohistochemistry and real-time reverse-transcription polymerase chain reaction in regenerated pulp tissue compared with normal pulp. Its migration effect was further examined in pulp CD105 + cells using small interfering RNA techniques to knock down the protein. Results Nine protein spots were detected solely in pulp CD105 + cells; one of these was identified as vimentin. The expression of vimentin messenger RNA was highest in pulp tissue among a variety of human tissues and higher in pulp CD105 + cells compared with other CD105 + cells and unfractionated total pulp cells. Pulp cells and endothelial cells were positively stained with vimentin in regenerated pulp tissue similarly as those in normal pulp tissue. The expression of vimentin in regenerated pulp was similar to normal pulp. RNA interference knock down of vimentin expression in pulp CD105 + cells significantly reduced the migration activity. Conclusions The highest expression of vimentin in pulp tissue among other tissues and its migration effect in pulp stem cells suggest that it is a quality standard for pulp regeneration and pulp cell function.

Pulp stem/progenitor cells can induce complete pulp regeneration. However, due to the limited availability of pulp tissue with age, there is a need to examine other sources for fractions of side population (SP) cells. In the present investigation bone marrow and adipose tissues of the same individual were evaluated as alternate sources. Pulp CD31(-) SP cells have higher migration activity and higher expression of angiogenic/neurotrophic factors than bone marrow and adipose CD31(-) SP cells. Adipose tissue CD31(-) SP cell transplantation yielded the same amount of regenerated tissue as pulp derived cells. However, bone marrow CD31(-) SP cell transplantation yielded significantly less regenerated tissue in pulpectomized root canals in dogs. The rate of matrix formation was much higher in adipose CD31(-) SP cell transplantation compared to pulp CD31(-) SP cell transplantation on day 28. Microarray analysis demonstrated similar qualitative and quantitative patterns of mRNA expression characteristic of pulp in the regenerated tissues from all three cell sources. Expression of many angiogenic/neurotrophic factors in the transplanted cells demonstrated trophic effects. Our results demonstrate that bone marrow and adipose CD31(-) SP cells might be suitable alternative cell sources for pulp regeneration.

The juxta-oral organ is a bilateral organ in the mammalian bucca. It consists of epithelial cords with surrounding mesenchyme. It develops from embryonic oral epithelium, but its macroscopic morphology in mice is less studied and seems to be very different from that of humans. The juxta-oral organ in mice extends more widely from the subcutaneous tissue of the mandible near the lateral fascia of the masseter to the submucosa of the soft palate. In this paper, we report that the mutant mouse allele Bmp7(lacZ) presented intense lacZ expression in the epithelial component of the juxta-oral organ in its homo- and heterozygous states. The main aims of this study were to show that this mutant mouse allele is suitable for observing macroscopic structure of the juxta-oral organ and to describe the development of this organ during embryonic and postnatal stages. Whole-mount beta-gal staining of this strain of mouse showed that the juxta-oral organ in mice appeared at E12.0 from oral epithelium and lost connection with it before E12.5. Then, the juxta-oral organ extended anteriorly to the lateral fascia of the masseter and posteriorly to the submucosal layer of the soft palate via the orbit. The mature juxta-oral organ had no connection to other epithelia such as those of the bucca and parotid duct. It persisted until adulthood and there seemed to be no tendency to regress. Transmission electron microscopy showed that each part of the juxta-oral organ was an epithelial cord surrounded by a basement membrane and mesenchymal tissue.

Aim: To achieve complete regeneration of dental pulp in vivo by stem/progenitor cells obtained from a fraction of side population (SP) cells from canine pulp. Materials & methods: A subfraction of SP cells, CD31-/CD146- SP cells, were isolated by flow cytometry from canine dental pulp. The efficiency of this subfraction of SP cells was evaluated in an experimental model of pulp injury in the dog. Results: The fractionated SP cells formed extensive networks of tube-like structures in vitro. Transplantation of the SP cells into an in vivo model of amputated pulp resulted in complete regeneration of pulp tissue with capillaries and neuronal cells within 14 days. Gene-expression studies demonstrated the expression of pro-angiogenic factors, implying trophic action on endothelial cells. Conclusions: This investigation demonstrates the potential utility of fractionated SP cells as a source of cells for total pulp regeneration complete with angiogenesis and vasculogenesis.

Gingipains, cysteine proteases derived from Porphyromonas gingivalis, are important virulence factors in periodontal diseases. We found that arginine-specific gingipain A (RgpA) increased the responsiveness of vascular endothelial cells to P. gingivalis lipopolysaccharides (LPS) and P. gingivalis whole cells to induce enhanced IL-8 production through protease-activated receptors (PARs) and phospholipase C (PLC) gamma. We therefore investigated whether RgpA-induced enhanced cell activation is mediated through exocytosis of Weibel-Palade bodies (WPBs) because they store vasoactive substances. RgpA rapidly activated PAR- and PLCgamma-dependent WPB exocytosis. In addition, angiopoietin (Ang)-2, a substance of WPB, enhanced IL-8 production by P. gingivalis LPS, suggesting that Ang-2 mediates the RgpA-induced enhanced cell responses. Thus, we propose a novel role for RgpA in induction of a proinflammatory event through PAR-mediated WPB exocytosis, which may be an important step for enhanced endothelial responses to P. gingivalis.

Osterix, a zinc-finger-containing transcription factor, is required for osteoblast differentiation and bone formation. Osterix is also expressed in dental mesenchymal cells of the tooth germ. However, transcriptional regulation by Osterix in tooth development is not clear. Genetic studies in osteogenesis place Osterix downstream of Runx2 (Runt-related 2). The expression of Osterix in odontoblasts overlaps with Runx3 during terminal differentiation in vivo. Runx3 down-regulates Osterix expression in mouse DPCs (dental pulp cells). Therefore the regulatory role of Runx3 on Osterix expression in tooth development was investigated. Enforced expression of Runx3 down-regulated the activity of the Osterix promoter in the human embryonic kidney 293 cell line. When the Runx3 responsive element on the Osterix promoter, located at −713 to −707 bp (site 3, AGTGGTT) relative to the cap site, was mutated, this down-regulation was abrogated. Furthermore, electrophoretic mobility-shift assay and chromatin immunoprecipitation assays in mouse DPCs demonstrated direct functional binding of Runx3 to the Osterix promoter. These results demonstrate the transcriptional regulation of Osterix expression by Runx3 during differentiation of dental pulp cells into odontoblasts during tooth development.

Genomic analysis of Mycoplasma pneumoniae revealed the existence of a large number of putative lipoprotein genes compared with the numbers in other bacteria. However, the pathogenic roles of M. pneumoniae lipoproteins are still obscure. In this study, we synthesized a lipopeptide (designated M. pneumoniae paralogous lipoprotein 1 [MPPL-1]) in which an S -dipalmitoylglyceryl cysteine was coupled to a peptide with a consensus sequence of a putative paralogous lipoprotein group characteristic of M. pneumoniae . The cytokine-inducing activity of MPPL-1 in human monocytic cells was much weaker (∼700-fold weaker) than that of the known mycoplasmal S -dipalmitoylated lipopeptide FSL-1 or MALP-2. MPPL-1 required Toll-like receptor (TLR2) to activate NF-κB-dependent gene transcription in HEK293 cells, although a 1,000-fold-larger amount of MPPL-1 was needed to exert activity similar to that of FSL-1 in the cells. TLR2-mediated recognition of MPPL-1 was synergistically upregulated by TLR6 but not by TLR1 or TLR10, although the activity was still weak. In addition, MPPL-1 did not antagonize FSL-1 recognition in human monocytic cells and TLR2/TLR6-expressing HEK293 cells. Thus, these results suggest that there is preferential selective recognition of diacylated lipopeptides due to the magnitude of an affinity with TLR2 and TLR6 and the roles of increased paralogous lipoprotein genes of M. pneumoniae in evasion of TLR2 recognition.

Introduction The proliferation and migration of dental pulp stem cells (DPSCs), a population comprised of dental pulp cells (DPCs), are important processes for pulp tissue repair. Dental pulp is exposed to changes in extracellular pH under various conditions, such as acidosis and exposure to caries-associated bacteria or a pulp capping agent. The objective of this study was to investigate the effects of extracellular pH on DPC proliferation and migration in vitro. Methods To evaluate the proliferation potency of DPCs in various extracellular pH conditions, 2 × 104 cells were seeded into 35-mm dishes. The following day, we changed to NaHCO3-free medium, which was adjusted to different extracellular pH levels. Results After 120 hours, DPCs cultured in media from a pH of 3.5 to 5.5 showed cell death, those cultured in conditions from a pH of 6.5 to 7.5 showed growth arrest or cell death, and those grown at a pH of 9.5 showed mild proliferation. The migratory activity of living DPCs was not affected by extracellular pH. For histologic analysis, human teeth possessing a small abscess in the coronal pulp chamber were sliced for histologic analysis. Proliferating cell nuclear antigen (PCNA) immunolocalization was used as an index of cell proliferation for the sections and cultured cells. Acidic extracellular pH conditions resulted in reduced numbers of PCNA-positive DPCs in the dishes. As for pulp tissue affected by a small abscess, a PCNA-negative pulp cell layer was observed in close proximity to the infectious lesion. Conclusions Together, these results suggest that an acidic extracellular pH condition is associated with DPC growth arrest or cell death.

Introduction This study was designed to evaluate the usefulness of magnetic resonance imaging (MRI) to assess the regeneration of pulp tissue. Methods Mobilized dental pulp stem cells and granulocyte colony-stimulating factor with collagen were transplanted into mature pulpectomized teeth for pulp regeneration (n = 4). The controls consisted of pulpectomized teeth with or without collagen and normal teeth with intact pulp tissue (n = 4, each). The signal intensity (SI) of MRI using T2 sequences was compared after the extraction of teeth in dogs. MRI was correlated with the corresponding histologic findings. Results Pulp tissue was fully regenerated 90 days after cell transplantation. On the other hand, the root canal was empty in the control collagen-transplanted teeth at 90 days. The SI of the normal teeth was significantly higher than that of nonvital pulpectomized teeth and the controls of collagen transplanted teeth at 90 days. The stem cell transplanted teeth showed a gradual decrease in the SI until 180 days at which time the SI was similar to that in the normal teeth and significantly higher than that in the teeth transplanted with collagen alone without the stem cells. Conclusions The changes in the SI of the pulplike tissue were consistent with the histologic findings, showing the potential usefulness of the noninvasive method to serially access the efficacy of pulp regenerative therapy.

Caries, pulpitis, and apical periodontitis increase health care costs and attendant loss of economic productivity. They ultimately result in premature tooth loss and therefore diminishing the quality of life. Advances in vital pulp therapy with pulp stem/progenitor cells might give impetus to regenerate dentin-pulp complex without the removal of the whole pulp. Tissue engineering is the science of design and manufacture of new tissues to replace lost parts because of diseases including cancer and trauma. The three key ingredients for tissue engineering are signals for morphogenesis, stem cells for responding to morphogens and the scaffold of extracellular matrix. In preclinical studies cell therapy and gene therapy have been developed for many tissues and organs such as bone, heart, liver, and kidney as a means of delivering growth factors, cytokines, or morphogens with stem/progenitor cells in a scaffold to the sites of tissue injury to accelerate and/or induce a natural biological regeneration. The pulp tissue contains stem/progenitor cells that potentially differentiate into odontoblasts in response to bone morphogenetic proteins (BMPs). There are two strategies to regenerate dentin. First, is in vivo therapy, where BMP proteins or BMP genes are directly applied to the exposed or amputated pulp. Second is ex vivo therapy and consists of isolation of stem/progenitor cells from pulp tissue, differentiation into odontoblasts with recombinant BMPs or BMP genes and finally transplanted autogenously to regenerate dentin. This review is focused on the recent progress in this area and discusses the barriers and challenges for clinical utility in endodontics.

The Sonic hedgehog (Shh)–Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61–64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1 -expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with ∼90% homology to that of GliH1 , indicating there is a family of GliH genes and raising the possibility of overlapping functions during development.