Periostin Is a Candidate Regulator of the Host Microenvironment in Regeneration of Pulp and Dentin Complex and Periodontal Ligament in Transplantation with Stem Cell-Conditioned Medium.

Purpose: The microenvironment is required for tissues to maintain their properties in vivo. This microenvironment encompasses the types and three-dimensional arrangement of cells forming the tissues, and their interactions with neighboring cells and extracellular matrices, as represented by the stem cell niche. Tissue regeneration depends not on the original tissue source of the transplanted cells, but on the microenvironment in which they are transplanted. We have previously reported pulp regeneration in a heterotopic root graft model by transplantation of conditioned medium alone, which suggests that host-derived cells expressing receptors for migration factors in conditioned medium migrate into the root canal and cause pulp regeneration. Regenerative medicine is needed to restore the original function of complex tissues. To achieve this, it is necessary to reproduce the changes in the microenvironment of the host tissue that accompany the regenerative response. Therefore, it is important to reproduce the microenvironment in vivo for further development of tissue regeneration therapy. Periostin is also found in the epithelial-mesenchymal junction, with expression sites that differ depending on the mineralized matrix stage, and is involved in regulation of calcification. Methods: We investigate whether periostin contributes to microenvironmental changes in regenerated pulp tissue. Dental pulp stem cells were induced into dentin, and gene expression of DSPP, nestin, DMP1, Runx2, and periostin was analyzed by qPCR and protein expression by IHC. Similarly, gene expression was analyzed using qPCR and protein expression using IHC in regenerated dental pulp obtained by ectopic transplantation. Results: Since these regenerated tissues were observable on the same slice, it was possible to understand changes in the microenvironment within the tissues. Conclusions: Periostin promoted proliferation of pulp stem cells, migration in type I collagen, and calcification in regenerated pulp, which strongly suggests that periostin is a promising candidate as a factor that contributes to the microenvironment of regenerated pulp.

Periapical bacterial disinfection is critical for dental pulp regenerative cell therapy in apical periodontitis in dogs.

BACKGROUND: Application of pulp regenerative cell therapy for mature teeth with periapical lesions is a critical clinical challenge. The bacterial infection in inaccessible location within the root canal system and in the periapical lesions could cause resistance and impediment, leading to limitations in successful therapy. Thus, the aim of this study was to examine the effect of residual bacteria on the outcome of pulp regeneration in mature teeth with apical periodontitis in dogs. METHODS: Periapical lesions were induced in 32 root canals of 4 dogs in two different models in severities, model A and model B. Model A (moderate infection): the canal exposed to the oral cavity for 2 weeks and then closed for 2 weeks. Model B (severe infection): the canal exposed to the oral cavity for 2 months and then closed for 5 months. All root canals were irrigated with 6% sodium hypochlorite, and 3% EDTA and further with 0.015% levofloxacin-containing nanobubbles, which was also used as an intracanal medicament. The aseptic conditions were examined by bacterial anaerobic culture and/or PCR analyses. The root canal treatment was repeated several times, and allogeneic dental pulp stem cells were transplanted into the root canals. The radiographic evaluation of periapical lesions was performed by cone-beam computed tomography before the first treatment, just after cell transplantation, and after 2 months and 6 months in both model A, model B, respectively. The animals were then sacrificed and the jaw blocks were harvested for histological and histobacteriological evaluations of pulp regeneration and periapical tissue healing. Furthermore, the DiI-labelled DPSCs were transplanted into the root canals after complete disinfection (n = 4) or without root canal treatment (n = 4) in the apical periodontitis model (model A) in one dog, and cell localization was compared 72 h after transplantation. RESULTS: In 8 out of 12 canals from model A, and 10 out of 15 canals from model B, pulp regeneration with good vascularization, innervation, and a significant reduction in the radiolucent area of the periapical lesions were observed. However, in the other 4 canals and 5 canals from model A and model B, respectively, no pulp tissue was regenerated, and inflammation in the periapical tissue, and external resorption or healed external resorption were detected. The presence of residual bacteria in the periapical tissues and severe inflammation were significantly associated with inhibition of regenerated pulp tissue in these 9 unsuccessful canals (P < 0.05, each) (OR = 0.075, each) analyzed by multiple logistic regression analysis. For cellular kinetics, transplanted cells remained in the disinfected root canals, while they were not detected in the infected root canals, suggesting their migration through the apical foramen under the influence of inflammation. CONCLUSIONS: A true pulp-dentin complex was regenerated in the root canal by the pulp regenerative therapy in mature teeth with apical lesions. The successful pulp regeneration was negatively associated both with residual bacteria and inflammation in the periapical tissue.

Age Related Senescence, Apoptosis, and Inflammation Profiles in Periodontal Ligament Cells from Canine Teeth.

OBJECTIVE: The periapical tissues, including periodontal ligament cells (PDLCs) play an important role in repairing the surrounding tissue of the teeth. A decrease in the regenerative potentiality of resident stem cells (PDLCs) has been suggested to be attributed to the decline of pulp regeneration. Therefore, examining the functional changes in periodontal tissue and cells that occur during the aging process is necessary. METHODS: The changes in the cementum extract (CE) and PDLCs isolated from young and aged dog teeth were evaluated. PDLCs growth rate, senescence markers, p16 and p21, and proinflammatory cytokines, IL-6, IL-1ß, and TNF-α, were analyzed by RT-PCR. Bax, an apoptosis marker, Bcl-2, a marker for cell survival, and IL-6 were examined by Western blot analyses to detect their variance expression in the CE. RESULTS: Our results demonstrated that aged PDLCs exhibit a low growth rate and an increased expression of p16; however, no change has been demonstrated in the expression of p21. The chronic inflammatory molecules, IL-6 and TNF-α, were significantly upregulated compared to young PDLCs. Western blot analyses showed decreased expression of Bcl-2 in the CE of the aged tooth (p < 0.001). CONCLUSION: Taken together, aging influences the functional changes of PDLCs and CE and increases senescence, chronic inflammation, and apoptosis markers. As a result, donor age is a key factor influencing the utilization of PDLCs for tooth regeneration.

Biological characteristics and pulp regeneration potential of stem cells from canine deciduous teeth compared with those of permanent teeth.

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

Pulp Regenerative Cell Therapy for Mature Molars: A Report of 2 Cases.

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.

Characterization of stable hypoxia-preconditioned dental pulp stem cells compared with mobilized dental pulp stem cells for application for pulp regenerative therapy.

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.

Effects of p-Cresol on Senescence, Survival, Inflammation, and Odontoblast Differentiation in Canine Dental Pulp Stem Cells.

Aging, defined by a decrease in the physical and functional integrity of the tissues, leads to age-associated degenerative diseases. There is a relation between aged dental pulp and the senescence of dental pulp stem cells (DPSCs). Therefore, it is important to investigate the molecular processes underlying the senescence of DPSCs to elucidate the dental pulp aging mechanisms. p-Cresol (PC), a uremic toxin, is strongly related to cellular senescence. Here, age-related phenotypic changes including senescence, apoptosis, inflammation, and declining odontoblast differentiation in PC-treated canine DPSCs were investigated. Under the PC condition, cellular senescence was induced by decreased proliferation capacity and increased cell size, senescence-associated ß-galactosidase (SA-ß-gal) activity, and senescence markers p21, IL-1ß, IL-8, and p53. Exposure to PC could stimulate inflammation by the increased expression of IL-6 and cause the distraction of the cell cycle by the increased level of Bax protein and decreased Bcl-2. The levels of odontoblast differentiation markers, dentin sialophosphoprotein (DSPP), dentin matrix protein 1, and osterix, were decreased. Consistent with those findings, the alizarin red staining, alkaline phosphatase, and DSPP protein level were decreased during the odontoblast differentiation process. Taken together, these findings indicate that PC could induce cellular senescence in DPSCs, which may demonstrate the changes in aging dental pulp.

Pulp Regeneration: Current Approaches, Challenges, and Novel Rejuvenating Strategies for an Aging Population.

We showed the safety and efficacy of pulp regenerative therapy by the autologous transplantation of mobilized dental pulp stem cells with granulocyte colony-stimulating factor in a pilot clinical study of young and middle-aged pulpectomized teeth. An experimental study in dogs further demonstrated an age-dependent decline in the amount of regenerated pulp tissue. In our society, in which people will soon live beyond 100 years, this therapy should be efficacious for contributing to the functional survival and endurance of the tooth not only for pulpectomized young teeth but also for aged teeth with periapical disease. However, there are 2 challenges: 1 is enhancing pulp regeneration in aged teeth, and another is complete disinfection before cell transplantation. Thus, this review presents trypsin pretreatment for the former and a novel irrigant, nanobubbles with antibacterial nanopolymers, for the latter, thus demonstrating potential utility for pulp regenerative therapy in aged teeth with periapical disease.

Treatment of Pulpectomized Teeth With Trypsin Prior to Transplantation of Mobilized Dental Pulp Stem Cells Enhances Pulp Regeneration in Aged Dogs.

There is an age-dependent decline of pulp regeneration, due to the decline of migration, proliferation, and cell survival of resident stem cells. Trypsin is a proteolytic enzyme clinically used for tissue repair. Here, we investigated the effects of trypsin pretreatment of pulpectomized teeth prior to cell transplantation on pulp regeneration in aged dogs. The amount of regenerated pulp was significantly higher in trypsin-pretreated teeth compared to untreated teeth. Trypsin pretreatment increased the number of cells attached to the dentinal wall that differentiated into odontoblast-like cells. The trypsin receptor, PAR2, was higher in vitro expression in the periodontal ligament cells (PDLCs) from aged dogs compared to those from young. The direct effects of trypsin on aged PDLCs were increased expression of genes related to immunomodulation, cell survival, and extracellular matrix degradation. To examine the indirect effects on microenvironment, highly extracted proteins from aged cementum were identified by proteomic analyses. Western blotting demonstrated that significantly increased fibronectin was released by the trypsin treatment of aged cementum compared to young cementum. The aged cementum extract (CE) and dentin extract (DE) by trypsin treatment increased angiogenesis, neurite extension and migration activities as elicited by fibronectin. Furthermore, the DE significantly increased the mRNA expression of immunomodulatory factors and pulp markers in the aged DPSCs. These results demonstrated the effects of trypsin on the microenvironment in addition to the resident cells including PDLCs in the aged teeth. In conclusion, the potential utility of trypsin pretreatment to stimulate pulp regeneration in aged teeth and the underlying mechanisms were demonstrated.

Immunomodulation and Regeneration Properties of Dental Pulp Stem Cells: A Potential Therapy to Treat Coronavirus Disease 2019.

The coronavirus disease 2019 (COVID-19) pandemic, originating from Wuhan, China, is known to cause severe acute respiratory symptoms. The occurrence of a cytokine storm in the lungs is a critical step in the disease pathogenesis, as it causes pathological lesions, pulmonary edema, and acute respiratory distress syndrome, potentially resulting in death. Currently, there is no effective treatment that targets the cytokine storm and helps regenerate the damaged tissue. Mesenchymal stem cells (MSCs) are known to act as anti-inflammatory/immunomodulatory candidates and activate endogenous regeneration. As a result, MSC therapy is a potential treatment approach for COVID-19. Intravenous injection of clinical-grade MSCs into COVID-19 patients can induce an immunomodulatory response along with improved lung function. Dental pulp stem cells (DPSCs) are considered a potential source of MSCs for immunomodulation, tissue regeneration, and clinical application. Although some current clinical trials have treated COVID-19 patients with DPSCs, this therapy has not been approved. Here, we review the potential use of DPSCs and their significance in the development of a therapy for COVID-19.

Nanobubble-Enhanced Antimicrobial Agents: A Promising Approach for Regenerative Endodontics.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

CCR3 antagonist protects against induced cellular senescence and promotes rejuvenation in periodontal ligament cells for stimulating pulp regeneration in the aged dog.

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-1ß. 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.

Animal Models for Stem Cell-Based Pulp Regeneration: Foundation for Human Clinical Applications.

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.

Magnetic resonance imaging in endodontics: a literature review.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Allogeneic transplantation of mobilized dental pulp stem cells with the mismatched dog leukocyte antigen type is safe and efficacious for total pulp regeneration.

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

Recent Progress in Translation from Bench to a Pilot Clinical Study on Total Pulp Regeneration.

Based on a preclinical bench study in dogs, a pilot clinical study was completed. Dental pulp stem cell (DPSC) subsets were isolated by mobilization by granulocyte colony-stimulating factor and expanded in good manufacturing practice conditions. The safety and efficacy of their autologous transplantation for total pulp regeneration was assessed in 5 patients with irreversible pulpitis. The quality control of the DPSC subsets was ensured by the absence of contamination and karyotype aberrations, and positive expression of stem cell markers. The clinical safety assessment was based on laboratory and radiographic evaluations, demonstrating no evidence of toxicity and adverse events. The efficacy was determined by the recovery of a sound positive response to the electric pulp test within 4 weeks and by the robust signal intensity of magnetic resonance imaging in the root canal at 24 weeks. The functional recovery of pulp tissue was determined by lateral mineralized tissue formation detected by cone beam computed tomography. This review presents a summary of the accumulating data in translation from bench to a pilot clinical study, demonstrating potential clinical utility of DPSC subsets for total pulp regeneration in endodontics.

Pulp regeneration by transplantation of dental pulp stem cells in pulpitis: a pilot clinical study.

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.

Mechanical forces induce odontoblastic differentiation of mesenchymal stem cells on three-dimensional biomimetic scaffolds.

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.

Assessment of Pulp Regeneration Induced by Stem Cell Therapy by Magnetic Resonance Imaging.

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.