Static magnetic field increases survival rate of dental pulp stem cells during DMSO-free cryopreservation

Abstract

Successful and efficient cryopreservation of living cells and organs is a key clinical application of regenerative medicine. Recently, magnetic cryopreservation has been reported for intact tooth banking and cryopreservation of dental tissue. The aim of this study was to assess the cryoprotective effects of static magnetic fields (SMFs) on human dental pulp stem cells (DPSCs) during cryopreservation. Human DPSCs isolated from extracted teeth were frozen with a 0.4-T or 0.8-T SMF and then stored at ?196?°C for 24?h. During freezing, the cells were suspended in freezing media containing with 0, 3 or 10% DMSO. After thawing, the changes in survival rate of the DPSCs were determined by flow cytometry. To understand the possible cryoprotective mechanisms of the SMF, the membrane fluidity of SMF-exposed DPSCs was tested. The results showed that when the freezing medium was DMSO-free, the survival rates of the thawed DPSCs increased 2- or 2.5-fold when the cells were exposed to 0.4-T or 0.8-T SMFs, respectively (p?<?0.01). In addition, after exposure to the 0.4-T SMF, the fluorescence anisotropy of the DPSCs increased significantly (p?<?0.01) in the hydrophilic region. These results show that SMF exposure improved DMSO-free cryopreservation. This phenomenon may be due to the improvement of membrane stability for resisting damage caused by ice crystals during the freezing procedure.     

Dental follicle progenitor cells responses to Porphyromonas gingivalis LPS

Periodontitis is a bacterially induced chronic inflammatory disease. Dental follicle progenitor cells (DFPCs) have been proposed as biological graft for periodontal regenerative therapies. The potential impact of bacterial toxins on DFPCs properties is still poorly understood. The aim of this study was to investigate whether DFPCs are able to sense and respond to lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major periopathogenic bacterium. Specifically, we hypothesized that LPS could influence the migratory capacity and IL‐6 secretion of DFPCs. DFPCs properties were compared to bone marrow mesenchymal stem cells (BMSCs), a well‐studied class of adult stem cells. The analysis by flow cytometry indicated that DFPCs, similar to BMSCs, expressed low levels of both toll‐like receptor (TLR) 2 and 4. The TLR4 mRNA expression was down‐regulated in response to LPS in both cell populations, while on protein level TLR4 was significantly up‐regulated on BMSCs. The TLR2 expression was not influenced by the LPS treatment in both DFPCs and BMSCs. The migratory efficacy of LPS‐treated DFPCs was evaluated by in vitro scratch wound assays and found to be significantly increased. Furthermore, we assayed the secretion of interleukin‐6 (IL‐6), a potent stimulator of cell migration. Interestingly, the levels of IL‐6 secretion of DFPCs and BMSCs remained unchanged after the LPS treatment. Taken together, these results suggest that DFPCs are able to sense and respond to P. gingivalis LPS. Our study provides new insights into understanding the physiological role of dental‐derived progenitor cells in sites of periodontal infection.     

Peculiar tooth renewal in a Jurassic ray-finned fish (Lepisosteiformes, †Scheenstia sp.)

Tooth replacement in vertebrates is extremely diverse, and its study in extinct taxa gives insights into the evolution of the different dental renewal modes. Based on μ-CT scans of a left lower jaw of the extinct fish †Scheenstia (Actinopterygii, Lepisosteiformes), we describe in detail a peculiar tooth replacement mode that is, as far as we could ascertain from the literature, unique among vertebrates. The formation of the replacement teeth comprises a 180° rotation of their acrodin cap that occurs intraosseously within bony crypts, and their setting up appears to be synchronous. We propose a model for the dental renewal process and identify complementary anatomical features visible in the tomography such as the junction between the different tooth-bearing bones (prearticular–coronoid and dentary), as well as cavities corresponding to intraosseous crypts, nervous and/or vascular canals. The location of the cavities and their subsequent identification (e.g. Meckel's cavity, mandibular sensory canal) help us to identify the function of pores visible on the bone surface and understand their relation to internal anatomical features. Finally, recognition of this tooth replacement mode raises the question of whether it is specific to †Scheenstia or related to a particular dentition type and thus potentially occurs in other lineages.     

Tea extracts differentially inhibit Streptococcus mutans and Streptococcus sobrinus biofilm colonization depending on the steeping temperature

Abstract

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping (p?<?0.05). Hot steeping significantly reduced bacterial growth (p?<?0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.     

On the release of fluoride from biofilm reservoirs during a cariogenic challenge: an in situ study

Abstract

Biofilm fluoride reservoirs may be a source of fluoride to the fluid phase during a sugar challenge reducing tooth mineral loss. However, the evidence for that is conflicting and has not been studied in biofilms containing different fluoride levels. In order to test fluoride release from biofilms with distinct fluoride concentrations, biofilms were grown in situ exposed to a combination of placebo, calcium and fluoride rinses forming biofilms with no (fluoride-free rinses), low (fluoride-only rinses) or high (calcium followed by fluoride rinses) fluoride concentrations, and collected before and 5?min after a sucrose challenge. Rinsing with fluoride increased fluoride concentration in the biofilm (p?<?0.05), mainly when a calcium pre-rinse was used before the fluoride (p?<?0.05). However, after a sugar challenge, no significant increase in the biofilm fluid fluoride concentration was observed, even in the fluoride-rich biofilms (p?>?0.05). Fluoride-rich biofilms do not release fluoride to the fluid phase during a sugar challenge.     

Ulex europaeus Agglutinin-I Binding to Dental Primary Afferent Projections in the Spinal Trigeminal Complex Combined with Double Immunolabeling of Substance P and GABA Elements Using Peroxidase and Colloidal Gold

Ulex europaeus agglutinin I (UEA-I) is a plant lectin with an affinity for L-fucosyl residues in the chains of lactoseries oligosaccharides associated with medium and smaller-diameter dorsal root ganglion neurons and their axonal processes. These enter Lissauer's tract and terminate within the superficial laminae of the spinal cord overlapping projections known to have a nociceptive function. This implies that the surface coatings of neuronal membranes may have a relationship with functional modalities. The present investigation further examined this concept by studying a neuronal projection with a nociceptive function to determine whether fucosyl-lactoseries residues were incorporated in its primary afferent terminals. Transganglionic transport of horseradish peroxidase (HRP) following injection into tooth pulp chambers was employed to demonstrate dental pulp terminals in the trigeminal spinal complex, while peroxidase and fluorescent tags were used concomitantly to stain for UEA-I. Double immunolabeling for substance P (SP) and γ-aminobutyric acid (GABA) using peroxidase and colloidal gold allowed a comparison of the distribution of a known excitatory nociceptive transmitter with that of UEA-I binding in specific subnuclei. Synaptic interrelationships between UEA-I positive dental pulp primary afferent inputs and specific inhibitory terminals were also examined.

SP immunoreactivity occurred in laminae I and outer lamina II (II_o) of subnucleus caudalis (Vc) and in the ventrolateral and lateral marginal region of the caudal half of subnucleus interpolaris (Vi), including the periobex area in which Vi is slightly overlapped on its lateral aspect by cellular elements of Vc. The adjacent interstitial nucleus (IN) also showed an intense immunoreactivity for this peptide antibody. UEA-I binding displayed a similar distribution pattern in both Vc and Vi, but extended into lamina II; and the superficial part of Lamina III in Vc. Dental pulp terminals were found to have a comparable distribution; however, many extended into the dorsal portion of the caudal half of Vi and the ventromedial quadrant of rostral Vi.

Electron-microscopic analysis showed that transganglionically labeled dental pulp terminals contained ovoid, complex membrane-bound vacuoles laden with transported HRP. The preterminal axon and synaptic membranes of those dental pulp terminals located in zones of Vc and Vi displaying an affinity for UEA-I were usually characterized by a patchy, electron-dense coating of the peroxidase tag. SP was demonstrated ultrastructurally with Protein-A colloidal gold (3-nm particles), whereas GABA immunoreactivity was revealed by the avidin—biotin—peroxidase method. This combined approach labeled a variety of simple axodendritic to large complex scalloped dental terminals which contained SP and were shown to have a UEA-I affinity. In addition, many of the larger terminals formed contacts with GABA-ergic dendrites and received inputs from GABA-ergic synapses. These complexes were most concentrated in lamina II_o of Vc and the ventrolateral zone of Vi. Many terminals in laminae II_i; and III with a UEA-I-positive surface coating failed to bind with the antiserum for SP, indicating that other transmitters may colocalize with UEA-I and suggesting that absolute correlations between specific oligosaccharide plasmalemmal coatings and functional modalities should be approached with caution. Further studies employing antisera to different transmitters are currently underway to better define the relationship between transmitter localization and anatomical substrates within this circuitry. These studies should eventually provide additional clues about relationships between functional properties and oligosaccharide coatings of primary afferent projections.