Dental properties, ultrastructure, and pulp cells associated with a novel DSPP mutation.

OBJECTIVE: To investigate physical characteristics and behaviours of dental pulp cells of teeth isolated from a dentinogenesis imperfecta (DGI) patient with a novel dentin sialophosphoprotein (DSPP) mutation. SUBJECTS AND METHODS: Whole exome and Sanger sequencing were employed to identify mutations. Physical characteristics of the teeth were examined. Pulp cells' behaviours including cell proliferation, colony-forming unit, osteogenic differentiation, pluripotent markers, and mesenchymal stem cell markers were investigated. RESULTS: The proband had opalescent brown primary teeth with extensive loss of enamel. Mutation analysis revealed a novel heterozygous 4-bp deletion, c.1915_1918delAAGT (p.K639QfsX674), in exon 5 of the DSPP associated with DGI. Analysis of the extracted primary incisor demonstrated a decrease in brightness but an increase in yellow and red chroma. The dentin showed reduced mineral density. The dentinal tubules were present in the predentin, but progressively collapsed in the dentin. The pulp cells exhibited markedly reduced CD105 expression, decreased cell proliferation, and smaller colony-forming units. CONCLUSIONS: We identified a novel mutation in the DSPP gene which disturbed dentin characteristics and pulp cells' behaviours. Our study expands the mutation spectrum and understanding of pathologic dentin phenotypes related to the frameshift deletion in the dentin phosphoprotein (DPP) region of the DSPP gene.

In Vitro Evaluation of Microleakage and Microhardness of Ethanolic Extracts of Propolis in Different Proportions Added to Glass Ionomer Cement.

OBJECTIVE: To evaluate the effect of ethanolic extracts of propolis (EEP) addition in different proportions to glass ionomer cement (GIC) on microleakage and microhardness of GIC. STUDY DESIGN: The cement was divided into four groups: one using the original composition and three with 10%, 25%, and 50% EEP added to the liquid and then manipulated. For microleakage assessment, sixty primary molars were randomly divided into four groups (n=15). Standard Class II cavities were prepared and then filled with EEP in different proportions added to GICs. Microleakage test was performed using a dye penetration method. The data were analyzed using one-way ANOVA and Mann-Whitney U tests (α = 0.05). Disc shaped specimens were prepared from the tested GIC to determine Vickers hardness (VHN). The data were analyzed using one-way ANOVA and post hoc Tukey test (α = 0.05). RESULTS: There were no statistically significant differences between the groups in terms of microleakage (p > 0.05). There were statistically significant differences between the VHN values of groups (p < 0.05). Increasing addition of EEP to GIC statistically significantly increased VHN value of GIC (p < 0.05). CONCLUSIONS: The addition of EEP to GIC increased the microhardness of the GIC and did not adversely affect the microleakage. Thus, it might be used during routine dental practice due to its antibacterial properties.

Nemotic human dental pulp fibroblasts promote human dental pulp stem cells migration.

Dental pulp inflammation has long been perceived as a negative factor leading to pulp disruption. Previous studies have suggested that the inflammatory reaction might be a prerequisite for the burst of progenitors implicated in pulp repair. To investigate the migration of human dental pulp stem cells (hDPSCs) in response to human dental pulp fibroblasts (HDPFs) nemosis, an in vitro model of nemosis-induced inflammation in three-dimensional culture was used in this study. We observed HDPF spheroid formation and that cell-cell adhesion between HDPFs leads to necrosis. Cell death detection and cell counting kit-8 assays showed reduced live cell numbers and increased levels of cell membrane leakage in HDPF spheroids. HDPFs spheroids expressed cyclooxygenase-2 and released an increasing amount of prostaglandin E2 and interleukin-8, indicating inflammation in response to nemosis. The Transwell assays showed that the conditioned medium from HDPFs spheroids significantly induced hDPSCs migration more than the medium from the monolayer. Taken together, these results indicate that HDPFs spheroids induce nemosis and contribute to the migration of hDPSCs. This model might provide a potential research tool for studying interactions between fibroblasts and stem cells, and studies concerning nemosis-targeted stem cells might help treat pulp inflammation.

Immunostaining of pulpal nerve fibre bundle/arteriole associations in ground serial sections of whole human teeth embedded in technovit® 9100.

A technique for embedding human undecalcified tooth specimens in Technovit® 9100 was developed, which permits immunohistological evaluation of pulp tissue in serial ground sections. Human molars were divided into 14-18 sections of about 23 µm thickness. Immunohistological double staining for S-100 and CD34 revealed unique associations of myelinated nerve fibre bundles with arterioles, which continued through the entire tooth pulp. These arterioles were not only accompanied by, but partially or totally enveloped in longitudinally orientated myelinated nerve fibre bundles. We speculate that this unique arrangement may mechanically support the arterioles and alleviate detection or regulation of their contraction state by sensory nerve cells.

Pulpal regeneration and root development after subcutaneous transplantation of cryopreserved immature teeth in rats.

The purpose of this in vivo study was to investigate revascularization and root growth after autotransplantation of cryopreserved immature teeth. Immature molar teeth were extracted in 4-week-old Wistar rats. In the test group, teeth were cryopreserved for 1 week and transplanted subcutaneously to the abdomen. In the control group, teeth were transplanted subcutaneously immediately after extraction. Material was collected in test and control animals at intervals of 1, 2, 4 and 10 weeks post-transplantation and histological and microradiographical examination was performed. Results showed that during the first weeks after transplantation, pulpal repair was similar in both groups although degenerated pulpal tissue was replaced slower in cryopreserved teeth and some differences in types of hard tissue formation were found between test and control teeth. After 10 weeks, the differences in the regenerated pulpal tissue between cryopreserved and control teeth observed during the first weeks were no longer detectable. No root growth was detected microradiographically 10 weeks after transplantation in any of the transplanted teeth. The presence of dentin-like tissue in the pulp cavity of some autotransplanted cryopreserved teeth, suggests survival of pulpal tissue after cryopreservation.

A comparison between two fourth generation apex locators.

AIM: The aim of this study was to evaluate ex vivo the accuracy of two fourth generation apex locators and compare the measurements obtained. METHODS: Forty single and multiroots of permanent teeth (i.e., sixty-two canals) without caries or restorations were selected. After determining the real canal length using a stereomicroscope and a digital calliper, we evaluated the accuracy of two fourth generation electronic apex locators (the Bingo 1020 and the Propex) by means of an experimental study model and an endodontic simulator. The experimental model uses a digital comparator to determine the root canal length to a precision of 0.001 mm, while the endodontic simulator replicates the normal clinical condition. The difference between the real length of each canal and that obtained with the two study models was calculated. RESULTS: In both experiments, the Bingo 1020 expressed a 94.35% and the Propex expressed a mean accuracy of 90.31% in positioning the file at ± 0.5 mm. CONCLUSION: The Bingo 1020 and the Propex apex locators are equally accurate and provide reliable measurements in calculating root canal length.

[Structural features of the pulp ground substance and its significance for acute and chronic pulpitis].

The goal of the research study is an analysis of amorphous material, fibers and cellular elements of the dental pulp and evaluation of their interactions with a variety of fibrouse structures in the norm and inflammation. To solve this problem used dental pulp tissue bioptats (10 cases) of patients with acute and chronic pulpitis and 10 control specimens (orthodontic operations). The material was studied by histological and electron microscopic methods of research. It was determined that in acute pulpitis develope changes promoting dissociation of fibrouse and cellular structures of pulp components, and thus, loss the cementing binding role of the ground substance. Acute pulpitis characterized by the recruitment of mast cells. ; The reorganization and remodeling of ground substance associated with neoangiogenesis, especially capillaries, and the replacement of collagen fibers by the fibrouse structures are major points in chronic pulpitis.

Effect of C-factor on microtensile bond strengths of low-shrinkage composites.

UNLABELLED: This study evaluated the microtensile bond strength (µ-TBS) of low-shrinkage composites with their corresponding adhesive systems, Filtek Silorane/Silorane adhesive (SIL, 3M ESPE AG, Seefeld, Germany) and Aelite LS/One-Step Plus (AL, BISCO Inc, Schaumburg, IL, USA) in cavities with different C-factors. Filtek Z250/Adper Single Bond Plus (Z, 3M ESPE, St Paul, MN, USA) was used as a control. METHOD: Standardized Class I cavities were prepared in extracted human molars after removing occlusal enamel. Cavities were assigned into six different C-factors by applying nail polish to four walls, three walls, two walls adjacent to each other, two walls opposite to each other, one wall, or no walls. Resin composites with their corresponding adhesive systems were applied according to manufacturer instructions. Specimens were sectioned to obtain four rectangular beams from the center of the restorations and µ-TBS was measured. Data were analyzed by Weibull survival analysis. Shrinkage stresses of the resin composites were determined after 30 minutes from the start of light-curing using a tensometer testing machine. Flexure elastic modulus was determined using standard procedures, in accordance with ISO 4049. Data for shrinkage stress and elastic modulus were analyzed by one-way analysis of variance followed by a Tukey multiple-comparisons test (p<0.05). RESULTS: µ-TBS of both SIL and AL were not affected by different C-factors; however, the bond strength of Z decreased significantly when the C-factor increased. Shrinkage stress results were 0.94 ± 0.1, 1.79 ± 0.18, and 2.14 ± 0.23 MPa for SIL, AL, and Z, respectively. The flexural modulus of both the SIL and the AL was significantly lower than that of Z. CONCLUSIONS: Increasing C-factor did not negatively affect the bond strength of low-shrinkage composites.

Regional odontodysplasia: management of an acute case with a scanning electron microscope.

Regional odontodysplasia (RO) is an uncommon, nonhereditary, odontogenic developmental disturbance characterized by hypoplasia and hypocalcification of the dental tissues that produce so-called "ghost teeth." This report describes a case of a 2.5-year-old girl who came to the clinic with RO affecting her right maxillary arch. The distinguishing characteristics of this case were the involvement of both the primary and permanent dentitions and the early occurrence of odontogenic abscesses that required the patient's hospitalization. Ultrastructural analysis revealed dental tissue failures that compromised the integrity of the involved teeth, justifying the high susceptibility to caries that was clinically observed. Follow-up was characterized by periodic prosthetic adjustments to maintain the patient's ability to masticate and for social interaction, beyond allowing normal development of her maxillofacial complex. Patients with RO require individualized treatment planning and close follow-up with a multidisciplinary approach.

LPS-induced autophagy in human dental pulp cells is associated with p38.

Lipopolysaccharides (LPS), which are components of the cell wall of Gram-negative bacteria, are among the important factors that induce inflammation, including pulpitis. Autophagy in human dental pulp cells (hDPCs) acts as a protective mechanism that promotes cell survival under adverse conditions through different signaling pathways. In this study, we examined whether LPS increases autophagy in hDPCs and investigated the role of mitogen-activated protein kinases signaling and nuclear factor κB (NF-κB) in this process. We found that stimulation of hDPCs with 0.1 µg/mL LPS increased the protein and mRNA levels of autophagy markers, beclin1 and microtubule associated protein light chain 3II (LC3II). In addition, acridine orange staining and transmission electron microscopy demonstrated the induction of autophagy upon the treatment of LPS. Furthermore, LPS affected phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and the nuclear translocation of NF-κB. While p38 inhibitor suppressed the LPS-induced increase in protein levels of beclin1 and LC3-II. Our results suggest that LPS induced autophagy in hDPCs and affected the phosphorylation of p38, ERK, and JNK, as well as the nuclear translocation of NF-κB. Phosphorylation of p38 may be involved in LPS-induced autophagy in hDPCs.

Isolation and in vitro characterisation of dental pulp stem cells from natal teeth.

Dental pulp stem cells were primarily derived from the pulp tissues of exfoliated deciduous teeth, primary incisors and permanent third molar teeth. The aim of this study was to isolate and extensively characterise SCs derived from human natal dental pulp (hNDP). For characterisation, proliferation capacity, phenotypic properties, ultrastructural and differentiation characteristics and gene expression profiles were utilised. A comparison was done between the properties of NDP-SCs and the properties of mesenchymal stem cells (MSCs) from bone marrow (BM) of the human. Stem cells isolated from hNDP and hBM were analysed by flow cytometry, reverse transcriptase-PCR, Real Time-PCR, and immunocytochemistry. Both cell lines were directionally differentiated towards adipogenic, osteogenic chondrogenic, myogenic and neurogenic lineages. hNDP-SCs and hBM-MSCs expressed CD13, CD44, CD90, CD146 and CD166, but not CD3, CD8, CD11b, CD14, CD15, CD19, CD33, CD34, CD45, CD117, and HLA-DR. Ultrastructural characteristics of hNDP-SCs showed more developed and metabolically active cells. hNDP-SCs and hBM-MSCs expressed some adipogenic (leptin, adipophilin and PPARgamma), myogenic (desmin, myogenin, myosinIIa, and alpha-SMA), neurogenic (gamma-enolase, MAP2a,b, c-fos, nestin, NF-H, NF-L, GFAP and betaIII tubulin), osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, and type I collagen) and chondrogenic (type II collagen, SOX9) markers without any stimulation towards differentiation under basal conditions. Embryonic stem cell markers Oct4, Rex-1, FoxD-3, Sox2, and Nanog were also identified. The differentiation potential of hNDP-SCs and hBM-MSCs to adipogenic, osteogenic, chondrogenic, myogenic and neurogenic was shown. This report described the first successful isolation and characterisation of hNDP-SCs.

Dens invaginatus: a qualitative-quantitative analysis. Case report of an upper second molar.

Dens invaginatus (D.I.) is a developmental anomaly caused by the infolding of the surface of a tooth crown before calcification has occurred. Its aetiology is controversial and remains unclear. It occurs in all dentitions with a prevalence that ranges from 0.25% to 7.74% and is mostly seen in the maxillary permanent incisors, particularly in the lateral incisors. Posterior teeth are infrequently involved. The purpose of this study was to investigate the morpho-structure of a second upper molar dens invaginatus compared with a control tooth. Ground and decalcified sections were prepared and histo-morphological evaluation of dental tissues was performed by using light microscopy, microradiography, and confocal laser scanning microscopy analysis (CLSM). The mechanical behaviour was tested by means of microhardness (HV) test. The results of our investigation showed structural anomalies of hard tissues, such as a difference in enamel prism diameter, in number and diameter of peripulpal dentinal tubules and in surface and diameter of cementocyte lacunae between D.I. and control tooth.

Scanning electron microscopy of pulp cavity dentin in dogs.

Dentin morphology and tubule diameter and density of peripulpal dentin were evaluated in 36 teeth from 12 adult dogs, aged between 2.5 and 13-years. The right maxillary canine and third premolar and right mandibular first molar teeth were extracted from euthanized dogs. The teeth were prepared and photomicrographs (n=108) were taken of the radicular and coronal dentin. Dentinal tubule density (tubules/mm2) was determined and tubular diameter and luminal area were measured in 3240 randomly chosen tubules using measurement software. Results from group 1 dogs (< 7-years-old) were compared with group 2 dogs (> 7-years-old). The majority of dentinal tubules were round or oval in shape and had uniform distribution at the radicular coronal third, and coronal levels. Dentin surfaces showed morphological differences at different levels of the tooth. Group 1 dentinal tubule diameter (1.87 +/- 0.44 microm) and area (1.91 +/- 0.83 microm2) were significantly different compared with Group 2 dentinal tubule diameter (1.53 +/- 0.39 microm) and area (1.22 +/- 0.50 microm2). There was no significant difference in tubular density between groups 1 (74,692 +/- 25,991 tubules/mm2) and 2 (72,938 +/- 24,646 tubules/mm2). Site-specific differences were observed in the pulp cavity dentin in the same tooth. These results provide a reference for future research in dogs or where dogs are used as a model for investigations in human dentistry.

Bond strength and adaptation of pulp capping materials to dentin.

This study evaluated the shear bond strength (SBS) and internal marginal adaptation of pulp-capping materials to dentin. Flat occlusal deep dentin surfaces were produced and randomly assigned to two groups (sound or artificial caries-affected dentin). The specimens in each group were assigned to one of seven subgroups according to the materials used: Biodentine, Theracal LC, Ultra-Blend plus, Calcimol LC, ApaCal ART, EQUIA Forte, and Ionoseal. Buildups (3-mm inner diameter and 2-mm deep) were made over the dentin surfaces. The bonded specimens were tested under shear forces at a crosshead speed of 0.8 mm/min and fracture modes were determined using a stereomicroscope at 25× magnification. The materials were applied to the pulp floor of prepared Class I cavities and then the cavities were restored with composite resin. Restored molar teeth were subjected to 5,000 thermocycles and sectioned in a bucco-lingual direction. Resin replicas were made to determine the adaptation at the pulp floor with scanning electron microscopy. Significant differences were determined among both bond strengths and gap formations of the materials. EQUIA Forte applied to both dentin substrates had a significantly higher SBS than the other materials. The bond strength of each material was not influenced by the dentin condition. Biodentine (3.03%), EQUIA Forte (7.83%), and Theracal LC (13.37%) had lower gap formations compared to other materials but were not significantly different from each other.

Characterization of Living Dental Pulp Cells in Direct Contact with Mineral Trioxide Aggregate.

Mineral trioxide aggregate (MTA) was introduced as a material for dental endodontic regenerative therapy. Here, we show the dynamics of living dental pulp cells in direct contact with an MTA disk. A red fluorescence protein (DsRed) was introduced into immortalized porcine dental pulp cells (PPU7) and cloned. DsRed-PPU7 cells were cultured on the MTA disk and cell proliferation, chemotaxis, the effects of growth factors and the gene expression of cells were investigated at the biological, histomorphological and genetic cell levels. Mineralized precipitates formed in the DsRed-PPU7 cells were characterized with crystal structural analysis. DsRed-PPU7 cells proliferated in the central part of the MTA disk until Day 6 and displayed a tendency to move to the outer circumference. Both transforming growth factor beta and bone morphogenetic protein promoted the proliferation and movement of DsRed-PPU7 cells and also enhanced the expression levels of odontoblastic gene differentiation markers. Mineralized precipitates formed in DsRed-PPU7 were composed of calcium and phosphate but its crystals were different in each position. Our investigation showed that DsRed-PPU7 cells in direct contact with the MTA disk could differentiate into odontoblasts by controlling cell-cell and cell-substrate interactions depending on cell adhesion and the surrounding environment of the MTA.

Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction.

Human dental pulp contains precursor cells termed dental pulp stem cells (DPSC) that show self-renewal and multilineage differentiation and also secrete multiple proangiogenic and antiapoptotic factors. To examine whether these cells could have therapeutic potential in the repair of myocardial infarction (MI), DPSC were infected with a retrovirus encoding the green fluorescent protein (GFP) and expanded ex vivo. Seven days after induction of myocardial infarction by coronary artery ligation, 1.5 x 10(6) GFP-DPSC were injected intramyocardially in nude rats. At 4 weeks, cell-treated animals showed an improvement in cardiac function, observed by percentage changes in anterior wall thickening left ventricular fractional area change, in parallel with a reduction in infarct size. No histologic evidence was seen of GFP+ endothelial cells, smooth muscle cells, or cardiac muscle cells within the infarct. However, angiogenesis was increased relative to control-treated animals. Taken together, these data suggest that DPSC could provide a novel alternative cell population for cardiac repair, at least in the setting of acute MI.

Effects of basic fibroblast growth factor on the development of the stem cell properties of human dental pulp cells.

We isolated adherent fibroblastic cells after collagenase and dispase treatment of human dental pulp. When human dental pulp cells (hDPCs) were cultured in the presence of basic fibroblast growth factor (bFGF), the ratio of hDPCs in the S-phase was significantly higher in comparison with incubation without bFGF. The ratio of hDPCs expressing STRO-1 as a marker of stem cell populations increased approximately eightfold in the presence of bFGF as opposed to that in the absence of bFGF. We demonstrated the characterization and distinctiveness of the hDPCs and showed that, when cultured with the medium containing serum and bFGF, they were highly proliferative and capable of differentiating in vitro into osteoblasts, chondrocytes, and adipocytes. Furthermore, the in vitro differentiation was confirmed at both the protein and gene expression levels. Transplantation of hDPCs -- expanded ex vivo in the presence of bFGF into immunocompromised mice -- revealed the formation of bone, cartilage, and adipose tissue. The donor hDPC-derived cells were labeled in the bone tissues located near the PLGA in the subcutaneous tissues of recipient mice using a human-specific Alu probe. When cultured with a serum-free medium containing bFGF, the hDPCs strongly expressed STRO-1 immunoreactive products and sustained self-renewal, and thus were almost identical in differentiation potential and proliferation activity to hDPCs cultured with the medium containing serum and bFGF. The present results suggest that the hDPCs cultured in the presence of bFGF irrespective of the presence or absence of the bovine serum are rich in mesenchymal stem cells or progenitor cells and useful for cell-based therapies to treat dental diseases.

SEM Study of apical morphological alterations in primary teeth with vital and necrotic pulps.

This study evaluated, by SEM, the morphology of human primary teeth roots. Twenty-four teeth were divided into 3 groups: pulp vitality (group I) and pulp necrosis without (group II) and with apical periodontitis (group III). Roots were analyzed by the presence of periodontal ligament (PDL) fibers and resorption areas. In groups I and II, presence of PDL fibers and absence of resorption were observed in all cases (100%), while all specimens (100%) of group III showed no PDL fibers and resorption areas. In conclusion, there are morphological differences in the apical region of primary teeth with different pulpal and periapical pathologies.

Artificial Caries Resistance in Enamel after Topical Fluoride Treatment and 445 nm Laser Irradiation.

OBJECTIVE: This in vitro study is aimed at investigating the caries preventive effectiveness of 445 nm diode laser in combination with topical fluoridation. MATERIALS AND METHODS: A total of 30 caries-free bovine teeth were used in this study. Eighteen teeth were covered with nail varnish except four windows on the labial surface. The windows were assigned to no treatment/control (C), laser (L) (0.3 W, 60 s, and 90 J/cm2), fluoride (F), and fluoride followed by laser (FL) treatment groups. Artificial caries lesions were created, and the teeth were sectioned and investigated under polarized light microscopy for quantitative measurement of the resulted lesion depth. Ten teeth were used for surface temperature measurement and two teeth for scanning electron microscopy (SEM). Extra twelve human molars were used for the intrapulpal temperature measurement. The absorbance of fluoride at 445 nm was measured. RESULTS: The means of lesion depth for the C, L, F, and FL groups were 123.48 (±21.93), 112.33 (±20.42), 99.58 (±30.68), and 89.03 (±30.38) µm, respectively. The pairwise differences of the L, F, and FL groups compared with the C group were significant (p < 0.05). The differences between groups were tested: FL versus L p=0.02, F versus L p=0.16, and FL versus F p=0.91, and the difference of the F versus FL was not significant (p=0.91). Temperature increment at the enamel surface and pulp roof were ∆T = 16.67 (±4.11) and 2.12 (±0.66)°C, respectively. The topical fluoride absorbance at 445 nm is five orders higher than that at 810 nm. SEM shows that after laser irradiation the enamel surface was intact and without thermal damage. CONCLUSIONS: The 445 nm laser irradiation may be useful for caries prevention, and its effectiveness is lower than those previously achieved using the argon ion laser.

The effect of hyaluronic acid hydrogels on dental pulp stem cells behavior.

Dental caries and trauma, particularly in childhood, are among the most prevalent teeth problems, which result in the creation of cavities and probably tooth loss. Thus, novel regenerative approaches with high efficiency and less toxicity are required. Stem cell therapy along with the implementation of scaffolds has provided excellent opportunities in the regeneration of teeth structure. Hyaluronic acid (HA) hydrogels have enticed great attention in the field of regenerative medicine. The unique chemical and structural properties of HA and its derivatives have enabled their application in tissue engineering. Several factors such as the location and type of the lesion, teeth age, the type of capping materials determine the success rate of pulp therapy. HA hydrogels have been considered as biocompatible and safe scaffold supports in human dental cell therapies.