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Seventy-two teeth in three Macaque fasicularis monkeys were used to evaluate pulpal response to cavit. Displacement of odontoblastic nuclei was evaluated histologically at three time periods in order to identify initial, early, and final responses. There were no statistically significant differences in pulpal response between Cavit and the zinc oxide-eugenol controls when used in accordance with the manufacturer's directions.
The pulps of Rhesus monkey teeth were exposed and capped with three materials: Teflon, a commercial hard-set calcium hydroxide (Ca(OH)2) material, and Ca(OH)2 plus saline. Experimental test periods were 3, 10, and 21 days, and 5 and 8 weeks. After treatment, the teeth were removed and processed for routine histologic evaluation. Teeth treated with the two Ca(OH)2 materials showed resolution of the inflammatory response and hard tissue formation at the exposure site as early as 10 days postoperatively, with consistent healing at 21 days and longer. Teflon had a similar soft tissue healing pattern but at a slower rate. Hard tissue formation at the exposure site in the teeth treated with Teflon was infrequent at the early time periods and present in only 20% of the teeth treated for 5 and 8 weeks. By evaluating the soft and hard tissue responses of the Ca(OH)2-capped and Teflon-capped teeth it may be possible, in future studies, to identify events unique to odontoblast differentiation during pulpal healing.
A case of an atypical odontogenic tumor with features of unicystic plexiform ameloblastoma and odontoblastic differentiation with deposition of tubular dentin matrix is reported. The significance of dentinogenesis, amelogenesis, and calcification in odontogenic tumors is discussed.
In order to assess the biocompatibility of dental materials, clinical studies, as well as in vitro studies, are needed. Although no bacteria were detected inside tubules, pulp reactions were observed when either adhesives of the last generation aiming to create an hybrid layer were used or restorative materials controlling the interface between the biomaterial and dental tissues. However, the reactions were feeble to moderate. Reparative dentine seldom appeared after such treatments. Differences between odontoblast and pulp cells, the nature of the lesion (necrosis or apoptosis), and differences between sound experimental and carious teeth seen in the real clinical situation are discussed in the context of in vivo human studies.
The occurrence of substance P (SP)-like immunoreactivity was studied in dental pulps of the cat. In untreated animals SP-positive fibres were found in all areas of the pulp. Most fibres were seen in central parts of the pulp but they were also observed in relation to the odontoblasts. Single, possibly unmyelinated, or fine caliber fibres or small bundles of them were seen running close to large non-fluorescent myelinated nerves, to blood vessels or without any obvious association with either of these structures. Fourteen days after transection of the inferior alveolar nerve no SP-positive fibres were observed in pulps on the denervated side. Transection of the cervical sympathetic ganglion did not change the occurrence of SP-positive fibres. The results indicate the existence of at least two types of afferent fibres in the dental pulp of the cat. Since the tooth pulp has been demonstrated to give rise only to pain sensation when stimulated, the results give morphological support for a role of SP neurones in pain transmission.
The following hypothesis is proposed: The inner enamel epithelium influences all cells of the dental papilla very early during embryologic development--long before any morphologic changes due to histo-differentiation are noticeable. During normal odontogenesis, the epithelium acts as a second order inducer for the differentiation of odontoblasts and their subsequent production of dentin. Other mechanisms exist which can induce responding fibroblasts of the dental pulp to produce dentin or dentin-like tissue. Collagen matrix can act as such an inducer.
The presence of enamel proteins, especially amelogenins, during root development has been a subject of controversy for a long time. Whereas some studies have reported the presence of enamel proteins on the root surface, others were not able to detect them at these places. Since microwave (MW) processing has been shown to improve the antigen retention in mineralised tissues, we have applied MW techniques to ultrastructurally analyse the presence of amelogenin during root formation in rat molars. Upper molar tooth germs from 12 and 13-day-old Wistar rats were fixed in 0.1% glutaraldehyde + 4% formaldehyde under MW irradiation. They were then decalcified in 4.13% EDTA, dehydrated in graded concentrations of ethanol and embedded in LR White Resin. Ultrathin sections were processed for post-embedding colloidal gold immunolabelling using a chicken egg yolk antibody against a 24 kDa rat amelogenin. Then, the grids were incubated with a rabbit anti-chicken IgG secondary antibody and with a protein A-gold complex. The immunoreactivity for 24-kDa amelogenin was detected in the cytoplasm of the epithelial diaphragm cells--the most apical portion of the Hertwig's epithelial root sheath (HERS), and in less amounts on the adjacent dental papilla extracellular matrix. Amelogenin was no longer observed at advanced stages of root dentinogenesis or later, during cementogenesis. The restricted presence of amelogenin at the early stages of root formation suggests that this protein may play a role in the differentiation of ectomesenchymal cells into root odontoblasts.
Amelogenin proteins comprise up to 90% of the organic matrix of developing enamel in the vertebrate tooth. Alternative splicing of mouse amelogenin pre-mRNA leads to the production of more than 14 protein isoforms, the functions of which are not totally understood. The smaller splice products, [A + 4] or M73 and [A - 4] or M59, have been shown to act differently as signaling molecules affecting odontogenic and other cell types. The mechanisms of these signaling processes, beginning with receptor identification, are not well understood. Utilizing radiolabeled [A - 4], we show here that 3H[A - 4] binds in a saturable fashion to the cell surface of C2C12 mouse fetal myoblasts at 4 degrees C, and not only binds at the surface but is internalized at 37 degrees C. "Far Western" immunohistochemistry performed on sections of E18 mouse incisors and molars with biotin-labeled [A - 4] as the primary ligand demonstrates [A - 4]-biotin binding to polarizing ameloblasts and odontoblasts, cells of the dental follicle, and along the stratum intermedium. Using [A - 4] affinity column chromatography and [A - 4]-biotin label transfer reaction, we have identified a 95 kDa C2C12 cell surface protein which bound [A - 4]. Utilizing Tandem MS (MS/MS) sequencing, we report the novel finding of the 95 kDa murine transmembrane protein, LAMP-1, originally identified as a lysosomal membrane protein that is also found at the cell surface, as an [A - 4] cell binding protein.
Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by inducing endothelial cell proliferation, migration and survival. Direct pulp capping with an adhesive resin system was shown to induce local increase in blood vessel density and lack of dentin bridging. However, the mechanisms involved in the increase in blood vessel density observed near the pulp exposures capped with an adhesive resin are largely unknown. OBJECTIVES.: To investigate the effect of an adhesive resin or one of its hydrophilic monomers (HEMA), in the expression of VEGF by pulp cells. METHODS.: Mouse odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), gingival fibroblasts, and macrophages were exposed to SingleBond (3M) or to 0-1000nM HEMA. VEGF expression was evaluated by ELISA and semi-quantitative RT-PCR. RESULTS AND SIGNIFICANCE.: VEGF expression was upregulated in MDPC-23 cells exposed to HEMA (p<0.001) or to SingleBond (p<0.018), and in macrophages exposed to HEMA (p<0.001) or SingleBond (p=0.001). In contrast, VEGF expression remained unchanged in undifferentiated pulp cells (OD-21), or fibroblasts exposed to either HEMA or Single Bond (p>0.05). Treatment with SingleBond or HEMA did not affect VEGF expression at the mRNA level of any cell type evaluated here, suggesting that the induction of VEGF expression in these cells is regulated primarily at the post-transcriptional level. These findings suggest that VEGF is involved in the regulation of pulp neovascularization observed in response to the application of adhesive resins at site of pulp exposure.
Previous studies show that the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), interferes with mineralization of the dental matrices in developing mouse and rat teeth. Culture of mouse embryonic molar teeth with TCDD leads to the failure of enamel to be deposited and dentin to undergo mineralization. Lactationally exposed rats show defectively matured enamel and retardation of dentin mineralization. To see if the impaired mineralization is associated with changes in the expression of dentin sialophosphoprotein (Dspp), Bono1 and/or matrix metalloproteinase-20 (MMP-20), thought to be involved in mineralization of the dental hard tissues, we cultured mouse (NMRI) E18 mandibular molars for 3, 5 or 7 days and exposed them to 1 microM TCDD after 2 days of culture. As detected by in situ hybridization of tissue sections, localization and intensity of Bono1 and MMP-20 expression showed no definite difference between the control and exposed tooth explants, suggesting that TCDD does not affect their expression. On the contrary, TCDD reduced or prevented the expression of Dspp in secretory odontoblasts and decreased it in presecretory ameloblasts. The results suggest that the retardation of dentin mineralization by TCDD in mouse molar teeth involves specific interference with Dspp expression.
The aim was to compare the use of different cell-material contact test methods with two different biological systems (cell line and tooth slice cultures) for cytotoxicity assessment of dental materials. Cytotoxicity of composites polymerized with two halogen-based and two light-emitting diode (LED) light-curing units (LCUs) served as the basis for comparison. Disk shaped specimens (7 x 2 mm) were fabricated using the four light sources. Composites were tested using L-929 cell line using direct/indirect/extract tests in accordance to standard protocols. Cytotoxicity was assessed using neutral red uptake. Tooth slice organ cultures were also employed to test the dental materials using direct/indirect test methods. Histomorphometric cell counting of intact odontoblasts and pulp fibroblasts and the use of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were applied for cytotoxicity evaluation. Discrepancy in result presentation was observed in the different tests used with L-929. Sensitivity levels of the L-929 tests ranked as follows: extract test < direct contact test < indirect contact test. Tooth slice tests confirmed that L-929 direct contact test proved to be the most reliable test among the three. In conclusion, this study highlights the risk involved when relying on a single test method for cytotoxicity assessment. It would be advisable to test different culture models and then proceed using more clinically relevant biological system that stimulate the in vivo situation for confirmation.
OBJECTIVE: The purpose of this study was to evaluate the pulpal response to an octacalcium phosphate (OCP)-based cement used as a pulp-capping material. STUDY DESIGN: The pulps of 60 maxillary first molars of male Sprague-Dawley rats were exposed and then capped directly by using either OCP-based cement or a calcium hydroxide slurry (control). Histologic examinations were performed at 1, 2, and 5 weeks after the surgical procedure, and the results were analyzed statistically by using the Mann-Whitney U test (P<.05). RESULTS: One week after pulp capping, the initial formation of reparative dentin in the exposed areas was more notable in the calcium hydroxide group than in the OCP-based cement group. At 2 weeks, reparative dentin covered by a layer of odontoblast-like cells was observed in both groups. However, at 5 weeks, reparative dentin consisting of regular dentinal tubules was observed more frequently in the OCP-based cement group. CONCLUSION: OCP-based cement allowed favorable healing processes to occur in the dental pulp.
Primary cultures of dental papilla-derived cells have a limited lifespan in vitro and can be maintained only up to passage 7-9 before showing senescence, but in vitro investigations often require a large number of cells showing phenotypic characteristics of the original tissue. To overcome this shortcoming, second-passage cells established from calf molar tooth germs by enzymatic pretreatment of the dental papilla were transfected by electroporation with pSV3neo, coding for the oncogene simian virus 40 large t antigen and a neomycin-resistance gene. Under selection by G418 (neomycin), four cell clones were isolated by single cell dilution at passage 15. Integration of simian virus 40 large t antigen and expression of the gene products were determined in cell clones by polymerase chain reaction (PCR) and immunohistochemistry. Four transfected cell lines (clones B, C, D and no. 12) were maintained in culture for over 1.5 years. For cell characterization, gene expression of procollagen alpha1 (I) and osteocalcin was evaluated by reverse transcriptase (RT)-PCR with cDNA obtained from the established cell lines at passage 20. Expression of collagen type I, osteocalcin and dentine phosphoprotein was evaluated immunohistochemically at passage 20 and after 1.5 years of continuous cell culture. Gene expression and the expression of mineralized tissue-specific proteins was demonstrated with RT-PCR and immunohistochemistry within all four immortalized cell lines. Expression of dentine phosphoprotein was observed in three simian virus 40 large t antigen-transfected cell lines, suggesting the immortalization of odontoblast-like cells in vitro. Thus, transfection of bovine dental papilla-derived cells resulted in immortal cell lines exhibiting phenotypic characteristics of the original tissue.
In response to injury, pulp precursor cells can differentiate into odontoblast-like cells that produce reparative dentine. In culture, pulp cells form mineralizing nodules, but the characteristics of the cells involved in this process are still not fully known. Human pulp cells for culture were obtained from coronal pulp isolated from non-erupted molars, and were maintained in RPMI 1640 medium supplemented with fetal calf serum. Nodules were forming in all human pulp primary cultures (HPPc) and human pulp subcultures observed until their fifth passage (HPSc<5). Mineralization of the nodules was confirmed by the presence of calcium and phosphate that were quantified by X-ray microanalysis. Specific immunolabeling revealed alpha-smooth muscle actin and vimentin in both HPPc and HPSc<5 cells. Cells positive for alpha-smooth muscle actin were either isolated or gathered together in the nodules. Under transmission electron microscopy, some cells in primary pulp cultures exhibited features typical of myofibroblasts or pericytes, such as stress fibers, fibronexus, indented nuclei and gap-junctions. These cells were frequently in close contact with mineral deposits. This work demonstrates for the first time the presence of pericytes or myofibroblasts in mineralized human pulp cultures, but further investigation is required to determine their origin, role and degree of differentiation.
This study sought to localize immunohistochemically Janus kinase (Jak) and Tyk isoforms and STAT-1 in association with events involved in early dentine and enamel formation in the rat molar. The Jaks and STATs (signal transducers and activators of transcription) are key signal-transduction pathway components in the cytokine receptor-linked pathway. The histological sections were not demineralized or fixed, providing optimum conditions for immunohistochemical localization. It appears that all of the Jak isoforms and STAT-1 are involved in enamel formation. Jak2 and STAT-1 colocalized in the proximal ends of presecretory and secretory-stage ameloblasts, supporting work by others that growth hormone receptor is located at that site. The colocalization of Jak1, Jak2 and STAT-1 along the proximal ends of presecretory and secretory ameloblasts suggests that the interferon receptor is up-regulated in these cells as well. Also, colocalization of Jak3 and STAT-1 in the proximal ends of the ameloblasts and the cells of the stratum intermedium predicts the location of the interleukin-7 receptor in those locations. Jak1, Tyk2 and STAT-1, but not Jak2 or Jak3, stain was seen in the odontoblasts.
The immunocalization of the small interstitial leucine-rich keratan sulphate proteoglycan lumican was studied in human teeth using a polyclonal antibody towards lumican and a standard indirect peroxidase technique. There was intense labelling for lumican in predentine, with immunopositivity also observed in and around the tubules, around the odontoblasts and in the pulp with a fibrillar distribution. The previous reported ability of lumican to control collagen fibrillogenesis and it putative function in collagen spacing suggest potential roles for lumican in predentine in relation to mineralization.
The murine S8 gene encodes a nuclear homeodomain containing transcription factor that is expressed at sites of epithelial-mesenchymal interactions, including those in cranofacial tissues. The spatiotemporal expression of S8 mRNA was examined in tooth primordia by in situ hybridization. S8 transcripts were found in all stages of tooth development in 13- to 16.5-day-old mouse embryos (E13-E16.5), covering the early bud stage up to the late bell stage. S8 mRNA was found exclusively in the ectomesenchyme and its derivatives that originate from the neural crest: future pulp cells, odontoblast precursors and dental follicle cells. Expression was highest at the late cap and early bud stages and declined at the mid-bell stage, in both first molar and incisor primordia. In E13 jaw explants grown in organ culture for 48 h, S8 mRNA was still present in first and second molar primordia after culture. At E15.5, S8 mRNA was also transiently present in the surrounding osteogenic tissue. It is concluded that the distribution pattern of S8 mRNA during tooth development indicates a role for the gene in defining the identity of dental papilla and follicle cells. It is speculated that the time-restricted expression of S8 in tooth primordia involves establishing the definitive form of the tooth organ.
The effect of this axotomy on the expression of vasoactive intestinal polypeptide (VIP) in trigeminal ganglion neurones and nerve fibres in the first molar was examined immunohistochemically 3 weeks postsurgically in rats. A distinct upregulation of VIP-like immunoreactivity was found in 3 to 4% of the neurones (mean dia., 20.9 +/- 0.45 microns; mean cross-sectional area, 367 +/- 13.2 microns 2) in the mandibular region after axotomy. An almost complete coexpression was registered in neurones upregulated for VIP and growth-associated protein 43. Coexpression of VIP and substance P (SP) was found in a small number of the immunoreactive (IR) small-sized neurones, mainly in those in which VIP and SP were both weakly immunoreactive. In the uninjured ganglion, weakly labelled VIP-positive granules were frequently traced at high magnification in the cytoplasm of small neurones. No VIP-IR fibres were found in the control molar pulp, except for a few in the root pulp near the apex. However, 3 weeks after axotomy, a number of VIP-containing nerve fibres were found in the molar pulp and apical periodontium. Coarse VIP-IR fibres directed towards the odontoblast layer were a common finding. In some of these fibres VIP was shown to colocalize with SP. These results indicate that VIP is clearly expressed and transported in regenerating primary afferent neurones after axotomy of the inferior alveolar nerve.