Imad About

Nestin expression in embryonic and adult human teeth under normal and pathological conditions.

Nestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. In the present study we examined the in vivo distribution of nestin in human teeth during embryonic development and in permanent teeth under normal and pathological conditions. The results show that nestin is first expressed at the bell stage and that its distribution is restricted in pulpal cells located at the cusp area of the fetal teeth. In young permanent teeth, nestin is found only in functional odontoblasts, which produce the hard tissue matrix of dentin. Expression is progressively down-regulated and nestin is absent from older permanent teeth. In carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions. In an in vitro assay system of human dental pulp explants, nestin is up-regulated after local application of bone morphogenic protein-4. A similar effect is seen in cultures of primary pulp cells during their differentiation into odontoblasts. Taken together, these results suggest that nestin plays a potential role in odontoblast differentiation during normal and pathological conditions and that bone morphogenic protein-4 is involved in nestin up-regulation.

Role of Human Pulp Fibroblasts in Angiogenesis

After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.

Apical leakage of four endodontic sealers.

The purpose of this study was to evaluate the sealing properties of four root canal sealers. Forty-eight maxillary central incisors were instrumented with Profile rotary instruments. They were randomly divided into four groups (n = 12) and filled using lateral condensation with one of the four sealers: Sealapex, Pulp Canal Sealer, AH 26, and Ketac-Endo. The apical leakage was measured with a fluid filtration method and expressed as L s(-1) KPa(-1). The teeth filled with Sealapex displayed a higher apical leakage (8.42 +/- 4.2 10(-11) L s(-1) KPa(-1)) than those filled with AH 26 (2.10 +/- 1.39 10(-11) L s(-1) KPa(-1)), Pulp Canal Sealer (0.17 +/- 0.09 10(-11) L s(-1) KPa(-1)) or Ketac-Endo (0.32 +/- 0.24 10(-1) L s(-1) KPa(-1)) (p < 0.01). No statistically significant difference was found among AH 26, Pulp Canal Sealer, and Ketac-Endo. No correlation was found between the sealing efficiency of the four sealers and their adhesive properties recorded in a previous study.

Factors influencing pulpal response to cavity restorations.

OBJECTIVES The purposes of this retrospective work were: (1) to determine the relative importance of bacteria on cavity walls, remaining dentin thickness and post-operative time on pulpal inflammation after cavity restoration; (2) to compare the respective influences of bacterial microleakage and the restorative material itself on pulp reaction severity. METHODS 317 class V cavities, in human bicuspids scheduled for extraction for orthodontic reasons were used for this study. Nine different materials were included. The severity of the pulpal reaction was ranked on hematoxylin/eosin stained sections according to FDI standards. The further parameters recorded were: (1) the presence or absence of bacteria on the cavity walls was noted on Brown and Brenn stained sections; (2) the remaining dentin thickness was measured and the teeth classified into three groups (< 500, 500-1000, > 1000 microns); and (3) the post-operative delay before extraction was recorded and classified as short time (< 5 weeks) or long time (> 5 weeks). Three two-way analyses of variance (ANOVA) followed by Kruskall and Wallis tests evaluated the influence of the three parameters on pulpal reaction severity. The third ANOVA also compared pulpal reactions under the different materials when the teeth were pooled, on bacteria free teeth and on bacteria contaminated teeth. RESULTS The first ANOVA ranked by decreasing order of importance: the presence of bacteria (p < 0.0001), the remaining dentin thickness (p = 0.02) and the post-operative delay (p = 0.04). The second ANOVA showed no difference among the restorative materials when bacteria were present on the cavity walls. SIGNIFICANCE The presence of bacteria on the cavity walls is the main factor influencing pulpal reaction under restorative materials, but does not account for 100% of the cases.

Cytotoxicity Testing of Endodontic Sealers: A New Method

The purpose of this study was to compare ISO standards versus a new technique for in vitro evaluation of cytotoxicity of root canal sealers. The cytotoxicity of AH Plus, Cortisomol, and Sealapex was first recorded according to ISO standards on L 929 fibroblasts by the MTT assay. In parallel, 30 single-rooted teeth were cut at the cementum enamel junction (CEJ), and the roots were prepared and sterilized before filling with the lateral condensation using one of three sealers (n = 10). The apexes of the roots were dipped into 1 ml of minimum essential medium for 1, 2, and 30 days renewing the medium every other day. After 24-h contact between the medium and the filled roots, the medium was used to measure the cytotoxicity on L 929 with the MTT assay. ISO standards always gave a statistically higher cytotoxicity than the root-dipping technique (p < 0.0001), whatever the sealer and the exposure time. The ISO standards showed statistically significant differences among the sealers (p < 0.0001). AH Plus was noncytotoxic, Cortisomol showed a high cytotoxicity decreasing over time (p < 0.001), and Sealapex displayed a high cytotoxicity that did not decrease over time (NS). The new technique showed statistically significant differences among the sealers (p = 0.001), but the differences were so small that they were likely not clinically relevant. The high cytotoxicity of Sealapex decreased over time but the cytotoxicity of AH Plus and Cortisomol did not. The results show that the ISO standards may strongly over-evaluate the cytotoxicity of the endodontic sealers, emphasize the difference among the sealers, and may clinically correspond to a large overfilling. The new technique reduces the discrimination of the test and may clinically correspond to a classical filling. Therefore, both methods might be considered as clinically relevant, corresponding to classical and overfilling conditions.

Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity

&NA; Odontoblasts form the outermost cellular layer of the dental pulp where they have been proposed to act as sensory receptor cells. Despite this suggestion, evidence supporting their direct role in mediating thermo‐sensation and nociception is lacking. Transient receptor potential (TRP) ion channels directly mediate nociceptive functions, but their functional expression in human odontoblasts has yet to be elucidated. In the present study, we have examined the molecular and functional expression of thermo‐sensitive TRP channels in cultured odontoblast‐like cells and in native human odontoblasts obtained from healthy wisdom teeth. PCR and western blotting confirmed gene and protein expression of TRPV1, TRPA1 and TRPM8 channels. Immunohistochemistry revealed that these channels were localised to odontoblast‐like cells as determined by double staining with dentin sialoprotein (DSP) antibody. In functional assays, agonists of TRPV1, TRPA1 and TRPM8 channels elicited [Ca2+]i transients that could be blocked by relevant antagonists. Application of hot and cold stimuli to the cells also evoked rises in [Ca2+]i which could be blocked by TRP‐channel antagonists. Using a gene silencing approached we further confirmed a role for TRPA1 in mediating noxious cold responses in odontoblasts. We conclude that human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth. Cultured and native human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth.

The effect of cavity restoration variables on odontoblast cell numbers and dental repair.

OBJECTIVES Dentinal repair following cavity restoration is dependent on several parameters including the numbers of surviving odontoblasts. The purpose of this study was to examine the effects of cavity cutting and restoration treatments on post-operative odontoblast numbers. METHODS 353 Standardised non-exposed rectangular Class V cavities, were cut into the buccal dentin of intact 1st or 2nd premolar teeth of 165 patients, aged between nine and 25 years of age. Composite cavity restorations with various etching treatments were compared with resin-modified glass ionomer cements, enamel bonding resins, as well as polycarboxylate, calcium hydroxide, and zinc oxide eugenol materials. Following tooth extraction (20-381 days) for orthodontic reasons, the area of the reactionary dentine and the area of the odontoblasts was measured histomorphometrically. RESULTS Odontoblast numbers and dentine repair activity were found to be influenced more by cavity restoration variables, than the choice of cavity filling materials or patient factors. The most important cavity preparation variable was the cavity remaining dentine thickness (RDT); below 0.25mm the numbers of odontoblasts decreased by 23%, and minimal reactionary dentine repair was observed. CONCLUSIONS Odontoblast injury increased as the cavity RDT decreased. In rank order of maintaining odontoblast numbers beneath restored cavities with a RDT below 0.5mm, and using calcium hydroxide for comparison; calcium hydroxide (100%), polycarboxylate (82.4%), zinc oxide eugenol (81.3%), composite (75.5%), enamel bonding resin (49.5%) and RMGIC (42.8%). The vitality and dentine repair capacity of the pulp is dependent on odontoblast survival. Variations in the extent of odontoblast injury caused during operative procedures, may be the major underlying reason for the success or failure of restorative treatments.

Human odontoblast cell numbers after dental injury

OBJECTIVES The purpose of this study was to measure the changes in odontoblast cell numbers in response to cavity restoration variables and patient factors, and the effect these factors have on dental repair by tertiary dentinogenesis. The number of vital odontoblasts is a critical factor for pulpal repair following restorative surgery, and yet little information is available on these cell numbers. METHODS Class V non-exposed cavities were prepared in the buccal surface of intact first or second premolar teeth of 27 patients, between 9 and 17 years of age. Following tooth extraction (28-163 days) the area of reactionary dentine and the area of the odontoblasts were measured histomorphometrically. RESULTS Patient factors, as well as cavity preparation and restoration variables, had little effect on the numbers of odontoblasts per pulpal unit area. However, the age of the patient did appear to have an effect on the reactionary dentine secretory capacity of odontoblasts per unit area, and on the relative number of odontoblasts beneath cut dentinal tubules. CONCLUSIONS Odontoblast cell numbers were maintained following the preparation of cavities cut into dentine with a 0.5mm residual dentine thickness. The repair capacity of the pulp-dentine complex would appear to be age dependent, this may explain differences in the success of various restorative treatments between patients.

Molecular Aspects of Tooth Pathogenesis and Repair: in vivo and in vitro Models:

Several growth factors and extracellular matrix molecules, which are expressed during embryonic tooth development, are re-expressed in dental tissues under pathological conditions. Pathological conditions such as caries lesions and dental injuries are often lethal to the odontoblasts, which are then replaced by other pulp cells. These cells are able to differentiate into odontoblast-like cells and produce a reparative dentin. Here we demonstrate the in vivo distribution of several molecules in human permanent teeth under normal and pathological conditions. The intermediate filament protein nestin, which is a marker of young odontoblasts, is absent from old permanent teeth. Similarly, the Notch protein, which is involved in cell fate specification and is localized in the sub-odontoblastic cell layer during odontogenesis, is not detected in adult dental tissues. In carious and injured teeth, nestin is expressed in a selective manner in odontoblasts surrounding the injury site, while Notch is expressed in the sub-odontoblastic layer of cells. We reproduced this physiological event in an in vitro culture system. Pulp cells cultured in the presence of P-glycerophosphate formed mineralization nodules. As odontoblasts, pulp cells contributing to the nodule formation express type I collagen, osteonectin, dentin sialophosphoprotein, and nestin. In this in vitro assay system, nestin is up-regulated after local application of Bone Morphogenetic Protein 2 and 4. Fourier transform infrared microspectroscopy showed that both the organic and the mineral compositions of the nodules have the characteristics of human dentin and differ from those of enamel and bone. These findings show that both the molecular and the mineral characteristics of the human dentin matrix are respected in the in vitro culture conditions.

E- and N-Cadherin Distribution in Developing and Functional Human Teeth under Normal and Pathological Conditions

Cadherins are calcium-dependent cell adhesion molecules involved in the regulation of various biological processes such as cell recognition, intercellular communication, cell fate, cell polarity, boundary formation, and morphogenesis. Although previous studies have shown E-cadherin expression during rodent or human odontogenesis, there is no equivalent study available on N-cadherin expression in dental tissues. Here we examined and compared the expression patterns of E- and N-cadherins in both embryonic and adult (healthy, injured, carious) human teeth. Both proteins were expressed in the developing teeth during the cap and bell stages. E-cadherin expression in dental epithelium followed an apical-coronal gradient that was opposite to that observed for N-cadherin. E-cadherin was distributed in proliferating cells of the inner and outer enamel epithelia but not in differentiated cells such as ameloblasts, whereas N-cadherin expression was up-regulated in differentiated epithelial cells. By contrast to E-cadherin, N-cadherin was also expressed in mesenchymal cells that differentiate into odontoblasts and produce the hard tissue matrix of dentin. Although N-cadherin was not detected in permanent intact teeth, it was re-expressed during dentin repair processes in odontoblasts surrounding carious or traumatic sites. Similarly, N-cadherin re-expression was seen in vitro, in cultured primary pulp cells that differentiate into odontoblast-like cells. Taken together these results suggest that E- and N-cadherins may play a role during human tooth development and, moreover, indicate that N-cadherin is important for odontoblast function in normal development and under pathological conditions.