EDTA conditioning of circumpulpal dentine induces dentinogenic events in pulpotomized miniature swine teeth.

AIM: To investigate pulp responses after pulpotomy and EDTA conditioning of pulp chamber dentinal walls with or without the placement of a collagenous scaffold in the experimental model of miniature swine teeth. METHODOLOGY: Forty-two fully developed permanent premolars and molars of healthy miniature swines were used. After preparation of pulp exposures through Class I cavities, the tissue of the pulp chamber was completely removed. The haemorrhage was controlled, and the root pulp was protected using a polyurethane film. The circumpulpal pulp chamber dentine was treated for 3 min with normal saline (group 1), or 17% EDTA solution (groups 2 and 3). The film was removed, and the pulp chamber cavities were left empty (groups 1 and 2), or filled with swine collagenous sponge (group 3). The access cavities were restored with a Teflon disc and glass ionomer. Teeth were evaluated histo-morphologically after 10 weeks. Data were compared using the nonparametric Fisher's exact test. RESULTS: Teeth after treatment of dentine with saline (group 1) were associated with no or only traces of hard tissue formation along the root canal walls. Atubular tertiary dentine deposition in the form of matrix deposition along root canal walls, or dentine bridge formation at the orifice of root canals or complete pulp canal obliteration, was found after treatment of dentine with EDTA in both experiments (groups 2 and 3). Significantly different types of mineralization in the root canals of groups 2 and 3 were seen (P = 0.001). Tissue changes in the pulp cavity, characterized by soft tissue growth and osteodentine or atubular tertiary dentine formation, were only seen after EDTA conditioning of dentine, in 6.2% of the teeth without scaffold and 64.7% of the teeth with scaffold application. Newly deposited mineralized matrix in the pulp chamber was always in continuation with hard tissue deposited in the root canals. CONCLUSIONS: The EDTA conditioning of pulp cavity dentinal walls after pulpotomy induced dentinogenic events in the root pulp. Application of collagenous scaffold in the pulp chamber enhanced soft tissue growth and mineralized tissue formation along the treated circumpulpal dentine.

Evaluation of Monomer Leaching from a Resin Cement Through Dentin.

The aim of the study was to evaluate the elution of Triethylene glycol dimethacrylate (TEGDMA), Urethane dimethacrylate (UDMA), Bisphenol A glycerolate dimethacrylate (BisGMA), and Bisphenol A (BPA), from a dual-cured resin cement through human dentin, under constant positive pulpal pressure. Ten human dentin disks were adjusted into a custom made testing device and transparent glass slabs were luted with Variolink II cement, under a steady pressure. The device was filled with Ringer's solution and a pressure of 14.1 cm H2O was applied. Eluates were retrieved from each one of the ten specimens at 9 time interval. All the samples were analyzed by High Performance Liquid Chromatography (HPLC). TEGDMA was detected from the second and UDMA was detected from the fourth time interval and then. The highest average concentration of TEGDMA and UDMA was detected in the 3 day time interval. Time had a significant effect on their elution. BPA and BisGMA were not detected in any sample of any time interval. The clinical relevance of the present study is that the concentration of the eluted monomers, under the conditions that were chosen, did not reach toxic levels for the pulp.

Transdentinal stimulation of tertiary dentine formation and intratubular mineralization by growth factors.

AIM: To evaluate the effects of recombinant growth factors on tertiary dentine formation and intratubular mineralization after their application on deep dentinal cavities in dog's teeth. METHODOLOGY: Treatment included dentinal etching (37% phosphoric acid) and applications of bioactive molecules (1 microg mL(-1) TGF-beta1, 10 microg mL(-1) IGF-1, 10 microg mL(-1) bFGF, 10 microg mL(-1) OP-1 or 1 microg mL(-1) monoclonal anti-human TGF-beta1 in phosphate buffered saline, PBS) at the dentinal base of buccal Class V cavities. Control groups were treated with 0.1% dog serum albumin (DSA) in PBS omitting the growth factors. This was performed both with and without dentinal etching. The dentinal responses regarding tertiary dentine formation and intratubular mineralization were assessed after 3 and 8 weeks, respectively, using light and scanning electron microscopy. Some specimens were also subjected to dentine permeability testing. RESULTS: The group treated with transforming growth factor-beta1 (TGF-beta1) and, to a lesser extent, the one treated with osteogenic protein-1 (OP-1) showed significantly greater (P < 0.05) tertiary dentine formation and intratubular mineralization over an 8-week period when compared with the control and the other experimental groups. There were no significant differences between groups in reduction in dentine permeability after treatment. CONCLUSION: Treatment of exposed dentinal tubules with biologically active molecules might induce intratubular mineralization and tertiary dentine formation. Further research is needed to substantiate any clinical benefits as opposed to traditional treatments of exposed dentine so as to provide a scientific base for the clinical regulation of dentine reactions.

Effect of simulated pulpal microcirculation on intrapulpal temperature changes following application of heat on tooth surfaces.

AIM: To evaluate ex vivo whether a simulated pulpal microcirculation inside a pulp chamber influenced intrapulpal temperature rise following application of heat on tooth surfaces. METHODOLOGY: An ex vivo model that allowed the circulation of 37 degrees C warm water inside the pulp chamber of an extracted human tooth was designed. The experimental model resembled pulpal microcirculation. After application of specific thermal stimuli for 30 s to the external surface of 15 maxillary central incisors, lateral incisors and canines, temperature changes were measured in the pulp chamber. The Greenhouse-Geisser and Bonferroni tests were used for analysis of the data. The level of significance was set at 0.05. RESULTS: Significant differences were found in all three groups of teeth between temperature measurements with or without intrapulpal water flow. Additionally, temperature changes resulting from the application of different stimuli to the group of lateral incisors were significantly greater compared with the other groups of teeth (P < 0.05). CONCLUSIONS: The importance of the cooling effect of simulated pulp microcirculation in the thermal behaviour of the dentine was established. Thickness of tooth tissue influenced significantly pulp temperature rise ex vivo.

Evaluation of monomer leaching from a resin cement through dentin by a novel model.

OBJECTIVE: To evaluate the elution of HEMA, BPA, UDMA and BisGMA from a conventional resin cement (Multilink Automix®, Ivoclar Vivadent) through human dentin, under constant positive pulpal pressure. METHODS: Ten human dentin disks (n=10) were adjusted in a new testing device and transparent glass slabs were luted with Multilink Automix® resin cement, following manufacturer's instructions, under a steady pressure of 25N. The device was filled with Ringer's solution. At 5min, 20min, 1h, 2h, 21h, 3 days, 7 days, 10days and 21days time intervals, the whole eluate was retrieved from each one of the ten specimens and then, the specimens were refilled with fresh Ringer's solution. The eluates were analyzed by High Performance Liquid Chromatography (HPLC). RESULTS: HEMA was detected in the eluate of all of the specimens, from 5min until 10 days. At four of the specimens, HEMA was also detected in the 21days eluate at very low concentrations. BPA, UDMA and BisGMA were not detected at any eluate. An unknown compound was also detected at 4.4min. SIGNIFICANCE: The concentrations of HEMA that enabled to diffuse from Multilink Automix® cement in an aqueous solution, through a dentin barrier, did not reach toxic levels and BPA, UDMA and BisGMA were not detected at all.

Study of the transdentinal diffusion of bioactive molecules.

In this work the mass transfer characteristics in a µ-tube that simulates a simplified dentinal tubule geometry are numerically investigated. The aim is to assess the key features that affect transdentinal diffusion of substances and consequently to define the necessary quantitative and qualitative issues related to a specific bioactive agent before its potential application in clinical practice. CFD simulations were performed in an S-shaped tapered micro-tube, while the code was validated using the non-intrusive optical measuring technique Laser Induced Fluorescence (LIF). As the phenomenon is one-dimensional, diffusion dominated and strongly dependent on the molecular size, the time needed for the concentration of released molecules to attain a required value can be controlled by their initial concentration. Thus, we propose a model, which is successfully verified by experimental data using a dentinal disc and which given the type of applied molecules and their critical pulpal concentration is able to estimate the initial concentration to be imposed.

Pulpal responses following direct pulp capping of healthy dog teeth with dentine adhesive systems.

OBJECTIVE: The aim of the present study was to evaluate the pulpal responses following direct pulp capping of mechanically exposed teeth with new dentine adhesive systems, in the preclinical model of dog teeth. METHODS: Class V cavities (approximately 2.50 mm wide, 3.00 mm long, 1.5-2.0 mm deep) were prepared on the buccal surface of permanent maxillary and mandibulary molars, two rooted premolars, canines and third incisors. The cavities were assigned to five experimental groups, representing one control group treated with a Ca(OH)2-based material and four experimental groups where the adhesive systems Clearfil SE Bond, Prompt-L-Pop, Etch & Prime 3.0 and Single Bond were tested. The pulpal tissue responses to dentine adhesives were assessed at post-operative periods of 7, 21, 65 days. RESULTS: Variable responses were recorded, which were characterized by moderate to severe inflammatory reactions, progressive extension of tissue necrosis with time and total absence of continuous hard tissue bridge formation after pulp capping with each of the four adhesive systems. Application of a Ca(OH)2-based material was characterized by inflammatory cell infiltration, limited tissue necrosis as well as partial to complete hard tissue bridging. CONCLUSIONS: It seems evident that application of dentine adhesive systems in direct contact with the mechanically exposed pulp of healthy dog teeth cannot lead to acceptable repair of the dentine-pulp complex e.g. wound healing with tertiary dentine bridge formation.

Compound maxillary odontoma in a young German shepherd dog.

A three-month-old, male German shepherd dog was admitted with a facial mass of two months' duration. Clinical examination showed a round mass, 3 cm in diameter, in the left infraorbital area. The upper last premolar deciduous tooth was not erupted. No other abnormalities were detected. Radiological examination revealed a posterior maxillary mass of mixed opacity. The mass was surgically excised. Histopathological examination demonstrated a connective tissue stroma containing foci of irregular enamel and dentine, resembling rudimentary teeth (denticles), surrounded by new bone formation. Morphology and structure of the denticles were also confirmed by scanning electron microscopy. A compound odontoma was diagnosed. One year after surgery, the dog was free of clinical signs.

Basic mechanisms of cytodifferentiation and dentinogenesis during dental pulp repair.

Based on recent literature, the specific potential of mature pulp cells to differentiate into polarized cells able to elaborate reparative dentin is described. These odontoblast-like cells are distinguished, by morphological criteria, from the other matrix-formative cells involved in non-specific defensive mechanisms of dental pulp. The suitable tissue conditions and the normal cascade of reparative events, allowing initiation of dentinogenesis in sites of amputated pulp, are presented. This is followed by a review of current observations on specific dentinogenic events, induced in various culture systems or in intrapulpal sites of mature teeth by artificial bio-molecules or bio-matrices. Data from these experiments are focused on the role of extracellular matrix molecules and growth factors in acquisition of the odontoblast-like cell phenotype and initiation of reparative dentinogenesis.

Inductive influences of demineralized dentin and bone matrix on pulp cells: an approach of secondary dentinogenesis.

The effects of demineralized dentin and bone matrix on dental ectomesenchymal cells were evaluated after observation periods of two or three weeks. Autogenous dentin and bone matrix, obtained from the crowns of primary molars or maxillary cortical bone, respectively, were demineralized with 3% acetic acid and implanted into pulpal or papilla sites of erupting dog teeth: first molars, fourth premolars, and canines. Dentin histogenesis associated with odontoblastic arrangement was demonstrated in relation to all dentin implants in pulpal sites. Deposition of osteodentin, followed in some areas by tubular predentin formation, was observed in contact with bone implants in pulpal sites. In papilla sites, the dentin implantation exhibited bone-like matrix formation, while bone implants were encapsulated by connective tissue. The interactions of pulp cells with demineralized dentin matrix constitute a model for experimental induction of secondary dentinogenesis and odontoblast-like cell differentiation.

Dentinogenic activity of allogenic plasma fibronectin on dog dental pulp.

The response of ectomesenchymal cells of dog dental pulp to implantation of Millipore filters supplemented with bovine plasma fibronectin was evaluated after observation periods of one or four weeks. Two concentrations of plasma fibronectin were used (0.2 and 1 mg/mL). Experiments also included implants treated with control solutions (PBS or 1 mg/mL of dog albumin). Formation of a layer of elongated, polarized cells was demonstrated in direct contact with the implants treated with 1 mg/mL of plasma fibronectin solution, after one week post-operatively. Microfilamentous organization and orientation of rough endoplasmic reticulum was observed mainly in the supranuclear zone of the polarized cells. Implants treated with the same solution were consistently surrounded by a thick layer of dentinal matrix after four weeks of their exposure to pulp sites. Implants treated with control solutions or with the low concentration of fibronectin never showed any sign of cell polarization and matrix synthesis. These data provide evidence that the pulp cells can express their odontoblastic phenotype in response to a surface containing concentrated fibronectin (even allogenic), without the need of other molecules as exogenous inductive factors.

Experimental bacterial anachoresis in dog dental pulps capped with calcium hydroxide.

The pulps of 36 permanent dog teeth were mechanically exposed and capped with Dycal, calcium hydroxide powder mixed with saline, or Teflon. At 2, 14, and 28 days postoperatively, nine teeth treated with the materials were extracted (treated control teeth): A suspension of streptococci was then injected intravenously. Twenty-four h later the dogs were killed and both the 27 treated teeth (experimental group) and 6 unoperated control teeth were removed in tissue blocks. Tissue sections were examined for the presence of bacteria, hard tissue formation, inflammatory cell response and necrosis. Bacteria were not observed in the unoperated and treated control teeth or in three of four teeth capped with Teflon for 29 days. In all the remaining specimens colonies of gram-positive cocci were found.

Formation of crystals on the surface of calcium hydroxide-containing materials in vitro.

The purpose of the present study was to examine the surface of calcium hydroxide-containing materials when treated in different in vitro conditions. Five calcium hydroxide-containing materials (Dycal, Nu-Cap, Life, Sealapex, and Apexit) and two control calcium hydroxide-free materials (Roth 811 and AH26) were tested. The materials were placed onto Teflon discs or root dentin samples; maintained in distilled water or phosphate-buffered saline, or culture medium supplemented or not supplemented with fetal calf serum; incubated at 37 degrees C in humidified atmosphere containing or not containing 5% CO2; and examined by scanning electron microscope. The results demonstrated precipitation of simple crystal units or organized crystalline structures in the calcium hydroxide-containing specimens treated in all experimental conditions, except those maintained in distilled water without 5% CO2. X-ray elemental microanalysis of the different crystalline structures showed one or two peaks corresponding to calcium or calcium and phosphorus. These data indicate that the crystals formed by reactions of calcium ions released from the calcium hydroxide-containing materials with the environmental ions might modify the material surface, especially in the presence of substrate adhesion molecules, such as fibronectin. This modification might play an important role in the regulation of cell adhesion and the initiation of new matrix synthesis.

Inductive effect of native dentin on the dentinogenic potential of adult dog teeth.

Autogenous dentinal matrix was exposed to the pulp cells of adult dogs in order to determine whether the mature pulp cells possess the ability to differentiate into odontoblast-like cells as a direct response to known inductive influences. The pulps of molars, premolars, and canines of three dogs (2 to 4 yr old) were mechanically exposed through buccal class V cavities. Pieces of demineralized or native dentin and predentin were implanted in the pulp sites for periods of 2 to 6 wk. The reactions were analyzed by light microscopy. Induction of dentin formation was observed only after native dentin implantation; either as early response to exposure of predentinal surfaces or around mineralized dentin after 3 postoperative wk. Encapsulation by fibrous connective tissue or matrix degradation was seen around demineralized dentin implants. A characteristic enhancement of circumferential pulpal dentin deposition around the implantation site was demonstrated after native dentin exposure to light and scanning electron microscopic examination. These data indicate that specific inductive influences given by the native but not the acid-conditioned dentin, when it is exposed to the pulp environment of adult teeth, are able to direct differentiation of odontoblast-like cells and to enhance the biosynthetic activity of primary odontoblasts.

A case report of a compound odontoma causing delayed eruption of a central maxillary incisor: clinical and microscopic evaluation.

A case of a compound odontoma caused delayed eruption of a central incisor in the maxilla is presented with clinical, radiographic, and microscopic findings. The odontoma was surgically removed and microscopic examination showed a lot of crown-like structures in a very irregular form, some of which were fused to each other at their apical parts. Enamel and pre-enamel were totally abnormal, whereas the inside of the pulp chamber tissue did not present any histological sign of functional tissue. The most homogeneous tissue was dentin. The removal of the odontoma was followed by a rapid eruption of the impacted central incisor.

Immunolocalization of fibronectin during the early response of dog dental pulp to demineralized dentine or calcium hydroxide-containing cement.

The role of fibronectin during the events initiating the post-developmental histogenesis of dentine was investigated by exposing the pulp to implants of autogenous demineralized dentine or calcium hydroxide-containing cement for short periods. Implants exposed for 3 days were processed for immunoelectron-microscopic analysis of fibronectin adsorption on to their surfaces. The localization of fibronectin in the critical area of interaction was examined by immunofluorescence 6, 14 and 21 days after implantation. Heavy adsorption of fibronectin on to the dentine implants and the crystalline structures that had been deposited on the cement implants was demonstrated. Positive fluorescence was consistently seen around dentine implants. Strongly immunopositive fibroblast-like cells and weakly reactive, differentiating odontoblast-like cells were found in association with the implanted matrix. Uncalcified matrix secreted by the polarized or non-polarized cells was consistently rich in fibronectin. Fibroblast-like cells exhibiting intense immunoreaction only at 14 and 21 days were mainly associated with the crystalline precipitates on the cement surfaces or within the surrounding pulp. The findings indicate that the specific inductive effects of demineralized dentine on pulp cells are initiated by exposure of the pulp to a fibronectin-containing surface; adhesion of pulp cells and synthesis of a fibronectin-rich matrix characterize the development of new dentine. The reparative response to non-specific inductive influences such as calcium hydroxide seems to be mediated by progressive enhancement of fibronectin synthesis in pulp cells.

Short-term dentinogenic response of dog dental pulp tissue after its induction by demineralized or native dentine, or predentine.

The events initiating the expression of odontoblastic potential by pulpal ectomesenchymal cells were investigated by exposing the pulp to demineralized, native and unmineralized autogenous dentine. The pulp responses to implants were histologically evaluated 3, 7 and 10 days postoperatively, while the surface structure of the newly mineralized matrices was examined 12 and 28 days after implantation. Differentiation of odontoblast-like cells in close proximity to the implanted matrix was consistently demonstrated after exposure to predentine. Scattered columnal cells undergoing polarization, characterized ultrastructurally by the orientation of their rough endoplasmic reticulum, were also found in direct contact with the demineralized dentine. However, in response to demineralized implants, groups of differentiated odontoblast-like cells were clearly seen only in association with a zone of matrix secreted in a polar, predentine-like pattern, indicating an asynchronous inductive influence of this type of implant on pulp cells. Further, the response of pulp cells to native dentine was characterized by the elaboration of a two-layered matrix (a fibrous and a polarly deposited matrix) before initiation of secondary dentinogenesis. Scanning electron microscopy of the newly deposited matrices revealed differences between the indirect matrix synthesis, observed in short-term response to implants of demineralized or native dentine, and the specific, dentinogenic function of the odontoblast-like cells. These observations indicate that the dentine-induced dentinogenesis is initiated by two mechanisms--direct induction of odontoblast-like cells as well as indirect matrix synthesis, which further controls cell polarization. Immobilization of the cells on implanted matrix seems to be the critical requirement for direct expression of the odontoblastic phenotype.

Effects of recombinant basic fibroblast growth factor, insulin-like growth factor-II and transforming growth factor-beta 1 on dog dental pulp cells in vivo.

The effects of recombinant basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-II and transforming growth factor (TGF)-beta 1 on dental pulp cells were investigated by light and transmission electron microscopy after their implantation for 1 and 3 weeks at central sites of mechanically exposed pulps in dog molar and canine teeth. The implants were Millipore filters that have been soaked with solutions containing 100 or 500 ng/ml of bFGF or IGF-II or 100 ng/ml of TGF-beta 1. Control filters were soaked with dog albumin. No changes in cell organization or matrix synthesis were seen after implantation of control filters. Groups of columnar, polarized cells with numerous mitochondria and Golgi elements or elongated cells unassociated with any matrix deposition were demonstrated after 1 or 3 weeks, respectively, in close proximity to the filters that had been soaked with bFGF solution; at a distance from these implants enhanced formation of an osteotypic matrix was seen beneath the exposure site. No particular response was found in close proximity to the filters that had been soaked with IGF-II solution after 1 or 3 weeks implantation but thick zones of osteodentine were found beneath the exposure site and at adjacent circumferential dentine sites. Numerous elongated, polarized cells with long cytoplasmic extensions invading the filter pores were consistently seen after 1 week in close proximity to the filters that had been soaked with TGF-beta 1 solution. After 3 weeks implantation of these filters, deposition of a tubular matrix surrounding the implants was seen in association with the highly elongated odontoblast-like cells, while enhancement of circumferential dentine formation was also found at adjacent peripheral sites. These experiments demonstrate that TGF-beta 1 when implanted for short term periods at central pulp sites exerted dentine-specific effects, inducing differentiation of odontoblast-like cells and stimulating primary odontoblasts. Implantation of bFGF and IGF-II did not result in reparative dentine formation, but did stimulate osteotypical matrix deposition at a distance from the implants.

Effects of glycosaminoglycans on in vitro mouse dental cells.

Trypsin-dissociated dental papillae and enamel organs removed from the first lower molars of day-18 and day-19 mouse embryos were cultivated for 2 to 4 days on Millipore filters in DMEM supplemented with 15 per cent fetal calf serum and chondroitin sulphate and hyaluronic acid, individually or together. Three concentrations of glycosaminoglycans (GAGs) were also added to the media (01, 0.2, 0.4 mg/ml). Control cultures were made in the absence of GAGs, and additional experiments performed in which the presence of GAGs was associated with serum-free medium. Elongated and polarized odontoblasts showing synthetic activity were only observed in the presence of serum containing medium supplemented with 0.1 or 0.2 mg/ml of the GAGs. [3H]-thymidine autoradiography demonstrated that these cells were already post-mitotic at the onset of the culture. Polarized ameloblasts were never observed. These data provide evidence that GAGs are able to maintain the polarized state of cultured odontoblasts.

Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentine matrix components.

The effects of dentine extracellular matrix components on dental mesenchymal cells were studied by light and transmission electron microscopy after their implantation at central sites of mechanically exposed pulps in dog molar teeth. The implants were Millipore filters that had been soaked with solutions containing 30 or 300 micrograms/ml of an EDTA-soluble fraction of rabbit incisor dentine. Control filters were soaked with dog albumin or phosphate buffered saline. Columnar, polarized cells were consistently seen after 8 days in close proximity to the filters coated with both concentrations of dentine matrix components. Characteristic features of these polarized cells included widened cisternae of the rough endoplasmic reticulum, a rich microfilamentous network in the long cytoplasmic extensions invading the filter pores and numerous cytoplasmic bodies. These cells also showed evidence of functional as well as cytological differentiation. Polarized processing of secretory granules could be observed after 8 days' implantation, and also the presence of matrix vesicles and deposition of a fine, collagenous matrix into the filters apically to the distal end of the cytoplasmic processes. After 24 days' implantation, secretion of a tubular matrix could be consistently seen in association with the odontoblast-like cells. No changes in cell organization or matrix synthesis were seen after implantation of control filters. These studies demonstrate that bioactive components present in the EDTA-soluble dentine matrix fraction are able to directly induce cell polarization and apical secretion of tubular matrix when implanted in contact with dental pulp cells at sites remote from the odontoblast layer.