N-Acetyl Cysteine Modulates the Inflammatory and Oxidative Stress Responses of Rescued Growth-Arrested Dental Pulp Microtissues Exposed to TEGDMA in ECM.

Dental pulp is exposed to resin monomers leaching from capping materials. Toxic doses of the monomer, triethyleneglycol dimethacrylate (TEGDMA), impact cell growth, enhance inflammatory and oxidative stress responses, and lead to tissue necrosis. A therapeutic agent is required to rescue growth-arrested tissues by continuing their development and modulating the exacerbated responses. The functionality of N-Acetyl Cysteine (NAC) as a treatment was assessed by employing a 3D dental pulp microtissue platform. Immortalized and primary microtissues developed and matured in the extracellular matrix (ECM). TEGDMA was introduced at various concentrations. NAC was administered simultaneously with TEGDMA, before or after monomer addition during the development and after the maturation stages of the microtissue. Spatial growth was validated by confocal microscopy and image processing. Levels of inflammatory (COX2, NLRP3, IL-8) and oxidative stress (GSH, Nrf2) markers were quantified by immunoassays. NAC treatments, in parallel with TEGDMA challenge or post-challenge, resumed the growth of the underdeveloped microtissues and protected mature microtissues from deterioration. Growth recovery correlated with the alleviation of both responses by decreasing significantly the intracellular and extracellular levels of the markers. Our 3D/ECM-based dental pulp platform is an efficient tool for drug rescue screening. NAC supports compromised microtissues development, and immunomodulates and maintains the oxidative balance.

Effect of lithium chloride on cell proliferation and osteogenic differentiation in stem cells from human exfoliated deciduous teeth.

Lithium Chloride (LiCl) has been used as a canonical Wnt pathway activator due to its ability to inhibit a glycogen synthase kinase-3. The aim of the present study was to investigate the effect of LiCl on cell proliferation and osteogenic differentiation in stem cells isolated from human exfoliated deciduous teeth (SHEDs). SHEDs were isolated and cultured in media supplemented with LiCl at 5, 10, or 20mM. The results demonstrated that LiCl significantly decreased SHEDs colony forming unit ability in a dose dependent manner. LiCl significantly enhanced the percentage of cells in the sub G0 phase, accompanied by a reduction of the percentage of cells in the G1 phase at day 3 and 7 after treatment. Further, LiCl markedly decreased OSX and DMP1 mRNA expression after treating SHEDs in an osteogenic induction medium for 7 days. In addition, no significant difference in alkaline phosphatase enzymatic activity or mineral deposition was found. Together, these results imply that LiCl influences SHEDs behavior.

Comparative Gene Expression Analysis of the Coronal Pulp and Apical Pulp Complex in Human Immature Teeth.

INTRODUCTION: This study determined the gene expression profiles of the human coronal pulp (CP) and apical pulp complex (APC) with the aim of explaining differences in their functions. METHODS: Total RNA was isolated from the CP and APC, and gene expression was analyzed using complementary DNA microarray technology. Gene ontology analysis was used to classify the biological function. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining were performed to verify microarray data. RESULTS: In the microarray analyses, expression increases of at least 2-fold were present in 125 genes in the APC and 139 genes in the CP out of a total of 33,297 genes. Gene ontology class processes found more genes related to immune responses, cell growth and maintenance, and cell adhesion in the APC, whereas transport and neurogenesis genes predominated in the CP. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining confirmed the microarray results, with DMP1, CALB1, and GABRB1 strongly expressed in the CP, whereas SMOC2, SHH, BARX1, CX3CR1, SPP1, COL XII, and LAMC2 were strongly expressed in the APC. CONCLUSIONS: The expression levels of genes related to dentin mineralization, neurogenesis, and neurotransmission are higher in the CP in human immature teeth, whereas those of immune-related and tooth development-related genes are higher in the APC.

Pulp development, repair, and regeneration: challenges of the transition from traditional dentistry to biologically based therapies.

The traditional concept of replacing diseased tooth/pulp tissues by inert materials (restoration) is being challenged by recent advances in pulp biology leading to regenerative strategies aiming at the generation of new vital tissue. New tissue formation in the pulp chamber can be observed after adequate infection control and the formation of a blood clot. However, differentiation of true odontoblasts is still more speculative, and the approach is largely limited to immature teeth with open apices. A more systematic approach may be provided by the adoption of the tissue engineering concepts of using matrices, suitable (stem) cells, and signaling molecules to direct tissue events. With these tools, pulplike constructs have already been generated in experimental animals. However, a number of challenges still remain for clinical translation of pulp regeneration (eg, the cell source [resident vs nonresident stem cells, the latter associated with cell-free approaches], mechanisms of odontoblast differentiation, the pulp environment, the role of infection and inflammation, dentin pretreatment to release fossilized signaling molecules from dentin, and the provision of suitable matrices). Transition as a process, defined by moving from one form of "normal" to another, is based not only on the progress of science but also on achieving change to established treatment concepts in daily practice. However, it is clear that the significant recent achievements in pulp biology are providing an exciting platform from which clinical translation of dental pulp regeneration can advance.

Characterization and gene expression of high conductance calcium-activated potassium channels displaying mechanosensitivity in human odontoblasts.

Odontoblasts form a layer of cells responsible for the dentin formation and possibly mediate early stages of sensory processing in teeth. Several classes of ion channels have previously been identified in the odontoblast or pulp cell membrane, and it is suspected that these channels assist in these events. This study was carried out to characterize the K(Ca) channels on odontoblasts fully differentiated in vitro using the patch clamp technique and to investigate the HSLO gene expression encoding the alpha-subunit of these channels on odontoblasts in vivo. In inside-out patches, K(Ca) channels were identified on the basis of their K(+) selectivity, conductance, voltage, and Ca(2+) dependence. In cell-attached patches, these channels were found to be activated by application of a negative pressure as well as an osmotic shock. By reverse transcription-polymerase chain reaction, a probe complementary to K(Ca) alpha-subunit mRNA was constructed and used for in situ hybridization on human dental pulp samples. Transcripts were expressed in the odontoblast layer. The use of antibodies showed that the K(Ca) channels were preferentially detected at the apical pole of the odontoblasts. These channels could be involved in mineralization processes. Their mechanosensitivity suggests that the fluid displacement within dentinal tubules could be transduced into electrical cell signals.

Effects of vitamin A deficiency on rat incisor formation.

Vitamin A deficiency (A-) is known to cause morphologic changes in tooth structures. However, its effects on glycosaminoglycan (GAG) distribution in dental pulp, and the role of retinoic acid (RA) in altering these effects are not clearly defined. Tissue changes induced by vitamin A deficiency and RA administration were evaluated histologically in incisors of rats fed on one of 3 different diets: a) vitamin A sufficient (A+); b) vitamin A deficient (A-); and c) vitamin A deficient supplemented with retinoic acid (A-/RA). Four weeks after the onset of vitamin A deficiency, all rats were killed and their 4 continuously erupting incisors evaluated histologically. A- rats had altered dentine and pulp with disrupted histodifferentiation of pulpal mesenchymal cells to normal odontoblasts. The frequency of these abnormalities in dentine and pulp was lower in A-/RA rats. The enamel organ was unremarkable in the 4-week deficient period. Using special stains, we noted that pulpal GAG accumulation in A- and A-/RA rats was limited to the lingual area, while in A+ rats, GAG were distributed throughout. These data suggest that vitamin A deficiency affects histodifferentiation of pulpal mesenchymal cells to odontoblasts, as well as GAG distribution in pulp. RA administration reduces the A- changes and therefore, appears to have some activity in dentinogenesis.