Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous Teeth Is a Feasible Stem Cell Resource for Regenerative Medicine

Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25–30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine.     

The presence of a side population and its marker ABCG2 in human deciduous dental pulp cells

Stem cells have been identified using the DNA-binding dye Hoechst 33342 and flow cytometry (FCM) in various tissues known as the side population (SP). The present study shows, for the first time, the presence of side population cells in human deciduous dental pulp cells (DPCs). Flow cytometric identification revealed that 2% of human deciduous DPCs were SP cells and that this SP profile disappeared in the presence of verapamil. The SP marker ABCG2 protein was localized to DPCs in the cell membrane by immunofluorescence staining, and flow cytometric analysis demonstrated that 3.6% of DPCs were ABCG2-positive. Furthermore, quantitative real-time PCR proved that ABCG2 mRNA expression in DPCs isolated from human exfoliated deciduous teeth was higher than in DPCs from permanent teeth. Our findings demonstrate that DPCs from human exfoliated deciduous teeth contain a higher proportion of the SP phenotype than permanent teeth and that they may constitute a stem cell population.     

Deciduous dental pulp stem cells are involved in osteoclastogenesis during physiologic root resorption

Multipotent mesenchymal stem cells are derived from the dental pulps of permanent teeth and exfoliated deciduous teeth, and are known to induce bone and dentin generation. However, the role of deciduous dental pulp stem cells (DDPSCs) in physiologic root resorption remains unclear. In this study, dental pulp stem cells (DPSCs) in permanent teeth (P) were retrieved and compared to DDPSCs from deciduous incisors at different root resorption stages: stable (S), middle (M), and final (F). Decalcified teeth sections showed that osteoclasts and resorption lacunae were most prevalent in the M resorption stage. DDPSC proliferation rate was also highest in the M stage. DDPSCs in the F stage produced more calcified nodules than those in the S or M stages. Alkaline phosphatase (ALP) expression was highest in the F stage, indicating that DDPSCs promote mineralization. In addition, the ratio of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) expression was significantly higher in the M stage, indicating that DDPSCs promote resorption. Dickkopf 1 (Dkk1) expression was remarkably higher in the F and P groups, suggesting that the Wnt pathway is inhibited during the resorption process. Interestingly, despite the fact that Wnt3a down‐regulated OPG in osteogenic induction medium and up‐regulated RANKL in medium with 1,25‐dihydroxy vitamin D3 (VD₃), the RANKL/OPG ratio was reduced only with VD₃. Collectively, our data indicate that DDPSCs influence osteoclastogenesis during the physiologic root resorption process, and that the canonical Wnt pathway can change the RANKL/OPG expression ratio in DDPSCs. J. Cell. Physiol. 228: 207–215, 2013. © 2012 Wiley Periodicals, Inc.     

Tooth-derived stem cells: Update and perspectives

Tissue engineering is an emerging field of science that focuses on creating suitable conditions for the regeneration of tissues. The basic components for tissue engineering involve an interactive triad of scaffolds, signaling molecules, and cells. In this context,stem cells(SCs) present the characteristics of selfrenewal and differentiation capacity, which make them promising candidates for tissue engineering. Although they present some common markers, such as cluster of differentiation(CD)105, CD146 and STRO-1, SCs derived from various tissues have different patterns in relation to proliferation, clonogenicity, and differentiation abilities in vitro and in vivo. Tooth-derived tissues have been proposed as an accessible source to obtain SCs with limited morbidity, and various tooth-derived SCs(TDSCs) have been isolated and characterized, such as dental pulp SCs, SCs from human exfoliated deciduous teeth, periodontal ligament SCs, dental follicle progenitor cells, SCs from apical papilla, and periodontal ligament of deciduous teeth SCs. However, heterogeneity among these populations has been observed, and the best method to select the most appropriate TDSCs for regeneration approaches has not yet been established. The objective of this review is to outline the current knowledge concerning the various types of TDSCs, and discuss the perspectives for their use in regenerative approaches.     

Talonid crests expression at the enamel–dentine junction of hominin lower permanent and deciduous molars

The application of microtomography (mCT) to dental morphological studies has unveiled a new source of palaeobiological information, particularly in the analysis of the internal structures of teeth. In this study, we assess the expression of talonid crests at the enamel and dentine surfaces in lower permanent and second deciduous molars (M₂ and dm₂) of H. sapiens, H. neanderthalensis and Atapuerca-Sima de los Huesos (SH) hominins. In modern humans, talonid crests are described exclusively in the deciduous teeth (Korenhof, 1982) and interpreted as a primitive mammalian remnant of the talonid attachment to the trigonid. Here we report for the first time the expression of talonid crests of deciduous and permanent molars in H. sapiens, H. neanderthalensis and Middle Pleistocene hominins. We discuss possible evolutionary interpretations and suggest the importance of recording this feature in future studies.     

Identification of novel epithelial stem cell-like cells in human deciduous dental pulp

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, ΔNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.     

年轻恒牙与成熟恒牙牙髓中ConA的表达

目的　研究刀豆素A (ConcanavalinA ,ConA)在人的年轻恒牙与成熟恒牙牙髓中的表达特征 ,以进一步了解牙髓发育成熟过程。方法　采用ABC法 ,对年轻恒牙和成熟恒牙牙髓标本进行ConA的免疫组化染色 ,并做图像定量分析。结果　ConA主要在牙髓成纤维细胞和成牙本质细胞的胞浆染色 ,同时在细胞外基质中可见阳性染色。年轻恒牙组呈强阳性染色 ,成熟恒牙组呈弱阳性染色。图像分析表明两组间灰度值有显著性差异 (P <0 0 1)。结论　年轻恒牙和成熟恒牙的牙髓组织中ConA的表达不同 ,提示可利用凝集素在组织细胞的表达情况来研究牙髓的分化、成熟状态。     

Viability of pulp stromal cells in cryopreserved deciduous teeth

The cryopreservation of exfoliated deciduous teeth and harvesting of stem cells from them as required would reduce the costs and efforts associated with banking stem cells from primary teeth. The aim of this study was determine whether the viability of pulp stromal cells from deciduous teeth was influenced by the cryopreservation process itself or the period of cryopreservation. In total, 126 deciduous teeth were divided into three groups: (1) fresh, (2) cryopreserved for <3 months (cryo<3), and (3) cryopreserved for 3–9 months (cryo3–9). The viability of the pulp tissues was compared among the three groups by evaluating the outgrowth from pulp tissues and cell activity within those pulp tissues. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) assay was performed to compare cell apoptosis within fresh pulp tissue and pulp tissue that had been cryopreserved for 4 months. The outgrowth from and cell activity within the pulp tissues did not differ significantly between the fresh and cryo<3 pulp tissues. However, these parameters were significantly reduced in the cryo3–9 pulp tissue. In TUNEL assay, 4-month cryopreserved pulp tissues has more apoptotic cells than fresh group. In conclusion, it is possible to acquire pulp stromal cells from cryopreserved deciduous teeth. However, as the period of cryopreservation becomes longer, it is difficult to get pulp cells due to reduced cell viability.     

Age-Dependent Impaired Neurogenic Differentiation Capacity of Dental Stem Cell is Associated with Wnt/β-Catenin Signaling

Two kinds of dental stem cells (DSCs), dental pulp stem cells (DPSCs) and stem cells from human-exfoliated deciduous teeth (SHED), have been identified as novel populations of mesenchymal stem cells that can be induced to differentiate into osteoblasts, chondrocytes, adipocytes, and neuron-like cells in vitro. As we know, both of them originate from the neural crest, but have distinct characteristics and functions in vitro and in vivo. The regeneration potential of DSCs declines with advanced age; however, the mechanism of the impaired potential in DSCs has not been fully explored. In this study, we investigated whether declined neurogenic differentiation capacity is associated with an altered expression of Wnt signaling-related proteins in vitro. We compared stem cells isolated from human dental pulp in two age groups: the exfoliated deciduous teeth (5–12 years), and the third permanent teeth (45–50 years). We found that the expression levels of neuron markers, such as βIII-tubulin, microtubule-associated protein 2(MAP2), tyrosine hydroxylase (TH), and Nestin were lower in the DPSCs group compared with that in the SHED group; however, in supplementation with human recombinant Wnt1 in the medium, the DPSCs were prone to neural differentiation and expressed higher levels of neurogenic markers. In summary, our study demonstrated that Wnt/β-catenin signaling may play a vital role in the age-dependent neural differentiation of DSCs. Therefore, DSCs may provide an ideal source of stem cells that can further extend their therapeutic application in nerve injury and neurodegenerative diseases.     

Critical revision of the alleged delayed dental eruption in South American “ungulates”

The endemic South American “ungulates” (SANU) were traditionally assumed to be a monophyletic offshoot of the Granorder Ungulata, but the current reorganization of the extant ungulates in Laurasiatheria and Afrotheria (based on molecular data) leaved them in an undetermined systematic position. The delayed dental eruption versus cranial growth was proposed as a hard-tissue synapomorphy of Afrotheria. In a recent paper, at least some endemic SANU (Notoungulata, Astrapotheria, and possibly Pyrotheria) were interpreted as allied to Afrotheres by having a late replacement of deciduous cheek teeth. This statement was based on: (1) the usual occurrence within these groups of individuals with deciduous and permanent teeth; (2) the individual size (estimated comparing the length/width ratio of cheek teeth) of specimens with permanent premolars erupted is indistinguishable from that of specimens with deciduous premolars (putative juveniles), and (3) the retention of at least dP1–dP3 in adult specimens of Parastrapotherium (Astrapotheria). Herein we critically examine the presumed existence of delayed dental eruption in astrapotheres, pyrotheres and xenungulates and the assumptions on which it was based. The alleged evidences supporting the occurrence of delayed dental eruption in SANU arise from misinterpreted information from the literature and conceptual mistakes (i.e. delayed dental eruption versus cranial growth was confused with delayed replacement of premolars versus molar eruption). Based on examination of at-hand specimens, we found that there is no evidence for a delayed premolar replacement relative to the eruption of the molars in astrapotheres, pyrotheres, and xenungulates. A delayed dental eruption in relation to jaw growth does not occur at least in Astrapotherium magnum. Although a very recent study proposed close relationships among afrotheres and at least notoungulates and xenungulates, a more complete analysis is still needed to elucidate the evolutionary relationships of astrapotheres and pyrotheres.     

The Study of Barium Concentration in Deciduous Teeth,Impacted Teeth,and Facial Bones of Polish Residents

The study determines the concentration of Ba in mineralized tissues of deciduous teeth, permanent impacted teeth, and facial bones. The study covers the population of children and adults (aged 6–78) living in an industrial area of Poland. Teeth were analyzed in whole, with no division into dentine and enamel. Facial bones and teeth were subjected to the following preparation: washing, drying, grinding in a porcelain mortar, sample weighing (about 0.2 g), and microwave mineralization with spectrally pure nitric acid. The aim of the study was to determinate the concentration of Ba in deciduous teeth, impacted permanent teeth, and facial bones. The concentration of barium in samples was determined over the ICP OES method. The Ba concentration in the tested bone tissues amounted to 2.2-15.5 μg/g (6.6 μg/g?±?3.9). The highest concentration of Ba was present in deciduous teeth (10.5 μg/g), followed by facial bones (5.2 μg/g), and impacted teeth (4.3 μg/g) (ANOVA Kruskal-Wallis rank test, p?=?0.0002). In bone tissue and impacted teeth, Ba concentration increased with age. In deciduous teeth, the level of Ba decreased with children’s age.     

Homologies of the anterior teeth in Indriidae and a functional basis for dental reduction in primates

In a recent paper Schwartz ('74) proposes revised homologies of the deciduous and permanent teeth in living lemuriform primates of the family Indriidae. However, new evidence provided by the deciduous dentition ofAvahi suggests that the traditional interpretations are correct, specifically: (1) the lateral teeth in the dental scraper of Indriidae are homologous with the incisors of Lemuridae and Lorisidae, not the canines; (2) the dental formula for the lower deciduous teeth of indriids is 2.1.3; (3) the dental formula for the lower permanent teeth of indriids is 2.0.2.3; and (4) decrease in number of incisors during primate evolution was usually in the sequence I3, then I2, then I1. It appears that dental reduction during primate evolution occurred at the ends of integrated incisor and cheek tooth units to minimize disruption of their functional integrity.