Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED)

Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness.     

Impaired neurite development associated with mitochondrial dysfunction in dopaminergic neurons differentiated from exfoliated deciduous tooth-derived pulp stem cells of children with autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restrictive interests, and repetitive stereotypic behaviors. Among the various mechanisms underlying the pathogenesis of ASD, dysfunctions of dopaminergic signaling and mitochondria have been hypothesized to explain the core symptoms of children with ASD. However, only a few studies focusing on the pathological association between dopaminergic neurons (DN) and mitochondria in ASD have been performed using patient-derived stem cells and in vitro differentiated neurons. Stem cells from human exfoliated deciduous teeth (SHED) are neural crest-derived mesenchymal stem cells present in the dental pulp of exfoliated deciduous teeth; these cells can differentiate into dopaminergic neurons (DN) in vitro. This study aimed to investigate the pathological association between development of DN and mitochondria in ASD by using SHED as a disease- or patient-specific cellular model. The SHED obtained from three children with ASD and three typically developing children were differentiated into DN, and the neurobiology of these cells was examined. The DN derived from children with ASD showed impaired neurite outgrowth and branching, associated with decreased mitochondrial membrane potential, ATP production, number of mitochondria within the neurites, amount of mitochondria per cell area and intracellular calcium level. In addition, impaired neurite outgrowth and branching of ASD-derived DN were not improved by brain-derived neurotrophic factor (BDNF), suggesting impairment of the BDNF signaling pathway in ASD. These results imply that intracerebral dopamine production may have decreased in these children. The earliest age at which deciduous teeth spontaneously exfoliate in humans, and SHED can be noninvasively collected, is approximately 6 years. Our results suggest that in vitro analysis of SHED-derived DN obtained from children with ASD provides neurobiological information that may be useful in determining treatment strategies in the early stages of ASD.     

Histological Techniques to Improve Testing of Pulpal Response of Teeth to Filling Material

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pu1p:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation. processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.     

Histological Techniques to Improve Testing of Pulpal Response of Teeth to Filling Material

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pu1p:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation. processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.     

Influence of chosen elements on the dynamics of the cariogenic process

This prospective study comprised 140 natural crowns of the teeth extracted from 31 boys and 35 men, as well as 39 girls and 35 women. They were divided into two groups. Group I consisted of primary teeth and group II consisted of permanent teeth. In each group, two subgroups were distinguished: subgroup A containing teeth without caries and subgroup B comprising carietic teeth. Zinc, iron, copper, nickel, chromium, cobalt, lead, cadmium, selenium, and strontium were determined in the samples by using the total reflection X-ray fluorescence method. Significantly higher concentrations of zinc, iron, copper, nickel, selenium, and strontium were detected in the crowns of healthy primary and permanent teeth than in the crowns of the carietic primary and permanent teeth. The concentrations of chromium, cobalt, lead, and cadmium were significantly higher in primary and permanent teeth with caries than in the healthy ones. Judging from the obtained results, we think that lower concentrations of zinc, iron, copper, nickel, selenium, and strontium together with higher concentrations of chromium, cobalt, lead, and cadmium in the carietic primary and permanent teeth, in relation with the respective concentrations of those elements in healthy teeth, can be one of the caries risk factors.     

Clinical grade adult stem cell banking

There has been a great deal of scientific interest recently generated by the potential therapeutic applications of adult stem cells in human care but there are several challenges regarding quality and safety in clinical applications and a number of these challenges relate to the processing and banking of these cells ex-vivo. As the number of clinical trials and the variety of adult cells used in regenerative therapy increases, safety remains a primary concern. This has inspired many nations to formulate guidelines and standards for the quality of stem cell collection, processing, testing, banking, packaging and distribution. Clinically applicable cryopreservation and banking of adult stem cells offers unique opportunities to advance the potential uses and widespread implementation of these cells in clinical applications. Most current cryopreservation protocols include animal serum proteins and potentially toxic cryoprotectant additives (CPAs) that prevent direct use of these cells in human therapeutic applications. Long term cryopreservation of adult stem cells under good manufacturing conditions using animal product free solutions is critical to the widespread clinical implementation of ex-vivo adult stem cell therapies. Furthermore, to avoid any potential cryoprotectant related complications, reduced CPA concentrations and efficient post-thaw washing to remove CPA are also desirable. The present review focuses on the current strategies and important aspects of adult stem cell banking for clinical applications. These include current good manufacturing practices (cGMPs), animal protein free freezing solutions, cryoprotectants, freezing & thawing protocols, viability assays, packaging and distribution. The importance and benefits of banking clinical grade adult stem cells are also discussed.     

Folic acid-mediated mitochondrial activation for protection against oxidative stress in human dental pulp stem cells derived from deciduous teeth

Enzymatic antioxidant systems, mainly involving mitochondria, are critical for minimizing the harmful effects of reactive oxygen species, and these systems are enhanced by interactions with nonenzymatic antioxidant nutrients. Because fetal growth requires extensive mitochondrial respiration, pregnant women and fetuses are at high risk of exposure to excessive reactive oxygen species. The enhancement of the antioxidant system, e.g., by nutritional management, is therefore critical for both the mother and fetus. Folic acid supplementation prevents homocysteine accumulation and epigenetic dysregulation associated with one-carbon metabolism. However, few studies have examined the antioxidant effects of folic acid for healthy pregnancy outcomes. The purpose of this study was to elucidate the association between the antioxidant effect of folic acid and mitochondria in undifferentiated cells during fetal growth. Neural crest-derived dental pulp stem cells of human exfoliated deciduous teeth were used as a model of undifferentiated cells in the fetus. Pyocyanin induced excessive reactive oxygen species, resulting in a decrease in cell growth and migration accompanied by mitochondrial fragmentation and inactivation in dental pulp stem cells. This damage was significantly improved by folic acid, along with decreased mitochondrial reactive oxygen species, PGC-1α upregulation, DRP1 downregulation, mitochondrial elongation, and increased ATP production. Folic acid may protect undifferentiated cells from oxidative damage by targeting mitochondrial activation. These results provide evidence for a new benefit of folic acid in pregnant women and fetuses.