Odontoblast Layer Research Articles

Sympathetic Regulation of Tertiary Dentinogenesis via Beta-2 Adrenergic Receptor on Rat Odontoblasts

IntroductionBeta-2 adrenergic receptor has been found within the osteoblast membrane meditating bone remodeling. Propranolol is a sympatholytic beta antagonist commonly used as long-term medication for the management of many common diseases such as hypertension. This study was performed to verify the presence of this receptor on odontoblasts in rats and, if present, to investigate its possible association with tertiary dentinogenesis. MethodsTwenty male Sprague-Dawley rats (9 weeks old) were randomly assigned to 4 groups: CP0.8 group, cavity preparation + propranolol treatment (0.8 mg/day, n = 5); CP4 group, cavity preparation + propranolol treatment (4.0 mg/day, n = 7); CON group, cavity preparation + saline treatment (0.2 mL/day, n = 5); and NT group, no treatment (n = 3). Cavity preparation was performed on the mesial aspect of the maxillary first molars bilaterally. After 2 weeks, the tertiary dentinogenesis (CP0.8, CP4, and CON) was examined by hematoxylin-eosin staining, and the localization of beta-2 adrenergic receptor (NT) was examined by immunohistochemistry. ResultsThe beta-2 adrenergic receptor immunoreactivity was observed in the odontoblastic layer in normal rat molar dental pulp. The tertiary dentinogenesis beneath the prepared cavity was significantly higher in the rats receiving 2-week systemic administration of propranolol than in those without the propranolol treatment. The higher-dose treatment of propranolol (P < .001) presented more effective up-regulation of tertiary dentinogenesis than the lower-dose treatment (P < .01). ConclusionsThese results indicate that the sympathetic nervous system decreases tertiary dentin formation via beta-2 adrenergic receptors located on rat odontoblasts. It suggests that adrenergic beta antagonist is expected to use in the treatment of inducing tertiary dentin formation to protect dental pulp.

Activation of the NLRP3/caspase-1 inflammasome in human dental pulp tissue and human dental pulp fibroblasts.

The NLRP3/caspase-1 inflammasome pathway plays an important role in cellular immune defence against bacterial infection; however, its function in human dental pulp tissue and human dental pulp fibroblasts remains poorly understood. We demonstrate that NLRP3 protein expression occurs to a greater extent in pulp tissue with irreversible pulpitis than in normal pulp tissue and in tissue with reversible pulpitis. Caspase-1 is present in its active (cleaved) form only in pulp tissue with irreversible pulpitis. NLRP3 and caspase-1 are expressed in the odontoblast layers in normal human dental pulp tissue, whereas in inflamed pulp tissue, the odontoblast layers are disrupted and dental pulp cells are positive for NLRP3 and caspase-1. Additionally, we investigate the role of the NLRP3/caspase-1 inflammasome pathway in human dental pulp fibroblasts and show that ATP activates the P2X7 receptor on the cell membrane triggering K+ efflux and inducing the gradual recruitment of the membrane pore pannexin-1. Extracellular lipopolysaccharide is able to penetrate the cytosol and activate NLRP3. Furthermore, the low intracellular K+ concentration in the cytosol triggers reactive oxygen species generation, which also induces the NLRP3 inflammasome. Thus, the NLRP3/caspase-1 pathway has a biological role in the innate immune response mounted by human dental pulp fibroblasts.

Response of Inflamed Pulps of Rat Molars after Capping with Pulp-capping Material Containing Fluocinolone Acetonide

IntroductionCapping inflamed dental pulp tissue is currently a controversial issue. To reduce pulp inflammation and stimulate pulp healing, a pulp-capping material containing fluocinolone acetonide (PCFA) has been developed. This study was aimed to evaluate the inflammatory response and hard tissue formation of inflamed dental pulps of rat maxillary molars after capping with Dycal, mineral trioxide aggregate (MTA), or PCFA. MethodsSixty maxillary rat molars were exposed to the oral environment for 48 hours. The exposed pulps were randomly divided into 6 groups (n = 10) according to pulp-capping materials (Dycal, MTA, or PCFA) and time (8 or 30 days). The cavities were capped and sealed with Fuji II LC. The animals were sacrificed after 8 and 30 days. Histologic specimens were prepared and evaluated for inflammatory response and hard tissue formation. ResultsEight days after pulp capping, all experimental groups showed disruption of the odontoblast layer in areas corresponding to the pulpal exposure. Acute inflammation was found in 80%, 60%, and 40% of samples in the Dycal, MTA, and PCFA groups, respectively. PCFA significantly decreased the pulp inflammation compared with Dycal. After 30 days, slight to moderate inflammation was found in all experimental groups. Hard tissue formation was found in 78%, 63%, and 100% of samples in the Dycal, MTA, and PCFA groups, respectively. No significant difference was found among the experimental groups. ConclusionsPulp capping with PCFA reduced the inflammation and stimulated hard tissue formation in the exposed pulps of rat molars. It may be used as a pulp-capping agent in inflamed pulps.

Leptin Promotes Dentin Sialophosphoprotein Expression in Human Dental Pulp

IntroductionLeptin, an inflammation-related adipokine, and its receptor (LEPR) are expressed in human dental pulp. Dentin sialophosphoprotein (DSPP) is involved in dentinogenesis and the dental pulp reparative response. The cell type expressing LEPR in dental human pulp and the resultant effect of the binding of leptin to LEPR remain unknown. This study describes the immmunohistochemical localization of LEPR and the effect of leptin on DSPP expression in human dental pulp. MethodsTwenty-five dental pulp specimens were obtained from freshly extracted caries-free and restoration-free human third molars. LEPR localization was examined by immunohistochemistry using the antihuman LEPR monoclonal antibody. The effect of leptin on DSPP expression was determined by immunoblot analysis and quantitative real-time polymerase chain reaction. ResultsImmunoreactivity for LEPR concentrated in the odontoblast layer but was not evident in the central zone of the dental pulp. Leptin dose dependently stimulated DSPP expression. Western blot analysis revealed the presence of a protein with an apparent molecular weight of ∼00 kDa, the estimated molecular weight of DSPP. The expression of DSPP messenger RNA was confirmed by quantitative real-time polymerase chain reaction, and the size of the amplified fragments (298 bp) was confirmed by agarose gel electrophoresis. ConclusionsThe present study shows that human dental pulp is immunoreactive for LEPR, with the immunoreactivity concentrated in the odontoblast layer, and that leptin stimulates, in a dose-dependent manner, DSPP protein and messenger RNA (mRNA) expression in human dental pulp. These findings further support the functional role of leptin in the dentin mineralization process and/or in dental pulp reparative and immune responses.

Predentin thickness analysis in developing and developed permanent teeth.

Background:Predentin, the unmineralized organic matrix is important in maintaining the integrity of dentin. It is usually thick where active dentinogenesis occurs. A wide variation in its thickness is reported. Hence, we determined the variation in predentin thickness at various sites of different age groups.Materials and Methods:60 freshly extracted teeth (maxillary and mandibular first premolars) were divided into three groups with 20 teeth in each as, Group 1 - teeth with incomplete root formation (age <16 years), Group 2 - teeth with complete root formation (aged between 16 and 30 years), Group 3 - teeth of patients aged above 30 years. The teeth were fixed, decalcified and sections of 6 μ thickness were obtained, and stained with hematoxylin and eosin. The distance between the odontoblastic cell layers of the pulp to the border line of the dentin was considered for the measurement of the predentin thickness. A total of nine sites were considered for each specimen.Results:The present study revealed varied mean predentin thickness at all nine sites in all three age groups. Maximum and minimum thickness was observed at the apex and pulp floor respectively in all three groups. There was a statistical significant difference in predentin thickness between groups 1 and 3 and 2 and 3.Conclusion:The predentin thickness in the first group gradually increased toward the growing end near the apex, while it was relatively constant in the second group and increased overall thickness at all the sites in the third group. A notable finding was a linear increase with age in width of the predentin and the thickness vary as a function of odontoblastic activity during different stages of tooth development.

The Influence of Liners on the Pulp Inflammation.

The study included application of liners and dental composites in to cavities of six experimental animals-rabbits (Oryctolagus cuniculus). OBJECTIVE The aim of the study was to investigate rabbit dental pulp response to different liners. Methods Cavity preparation for class V were made on the maxillary central incisors and one lower incisor, while the second lower incisor served as a control tooth. These teeth were restored with the use of one of the following liners--Calcimol LC, ANA Liner and Fuji II LC Improved, and Ceram-X mono dental composite. After an observation period of five days animals were sacrificed and prepared for histological analysis.The existence and degree of the pulp inflammation was determined by using a light microscope. Results showed that the used liners do not cause distortion of the structure and continuity of the odontoblastic layer. Inflammation was not registered in the control group, while in each group of tested materials one tooth with mild signs of hyperemia was registered. Results showed that all three tested liners demonstrated favorable effects on the pulp of the tooth and did not lead to inflammatory reactions. Histological analysis of the dental pulp of experimental animals suggests that the liners used in this study do not compromise the integrity of the odontoblastic layer, if it is applied over a thin layer of dentin. In each group of tested materials one tooth with mild signs of hyperemia and vasodilation was registered.

Driving the Mineral out Faster: Simple Modifications of the Decalcification Technique

Quicker decalcification is essential for faster diagnosis of hard tissue pathology. Heat and mechanical agitation are known to hasten decalcification. To compare the rate of decalcification, cellular and staining characteristics of decalcified specimens of bone and teeth by using the conventional method (10% formal formic acid), heating to 45(o)C and by physical agitation with magnetic stirrer. Weight-matched samples of caprine-origin bone (n=15) and teeth (n=15) were decalcified using three methods namely: a) Gooding and Stewart's fluid; b) Gooding and Stewart's fluid heated to 45(o)C for 6 hours daily; and c) Gooding and Stewart's fluid agitated using a magnetic stirrer for 6 hours daily. Non-lesional skin tissue samples were placed along with each specimen. End point of decalcification (chemical test) was noted; 4 micron sections were taken and stained with H&E. Differences in rate of decalcification and staining characteristics were assessed by Kruskal Wallis test and chi-square test respectively. Hard tissues decalcified faster with stirring and heating methods. The amount of osteocyte retraction noted in bone was significantly reduced in the stirring method. In tooth specimens, modified techniques resulted in poorer nuclear-cytoplasmic contrast of pulp cells. Heating affected the odontoblast layer. Soft tissues exhibited higher eosinophilia in stirring and conventional methods, whereas nuclear-cytoplasmic contrast and chromatin staining was poorest in heating and conventional methods. Physical agitation of decalcifying fluid may be recommended while maintaining satisfactory quality of tissue morphology and staining.

Insulin-like Growth Factor Expression in Malnourished Rat Dental Pulp

This study evaluated the expression levels of IGF-I, insulin peptides, and their respective receptors, IGF-IR and IR, in the dental pulp of rats subjected to protein malnutrition. The nourished group (N) received a diet with 20% casein, while the malnourished group (M) received a diet with 5% casein. The renourished group (R) consisted of M group members maintained on the N group diet from the 22nd to the 60th day of life to evaluate the effects of renourishment. Dental pulps of the maxillary first molars were stained with hematoxylin and eosin, and picrosirius red, and were subjected to immunohistochemical tests. Malnutrition altered the odontoblastic layer and the collagen component of the dental pulp. IGF-I and IGF-IR expression levels were higher in the N group but there were no differences in the insulin expression levels between the groups. IR immunoexpression was higher in the N group compared with groups M and R. The results suggest that protein deprivation changed the constitution of the dental pulp and affected the factors that influence its development. The process of renutrition during puberty was not able to support the recovery of pulp morphology.

Wnt5a regulates dental follicle stem/progenitor cells of the periodontium.

IntroductionDental follicle gives rise to one or several tissues of the periodontium including the periodontal ligament, cementum and/or alveolar bone. Whether Wnt5a is expressed in the postnatal periodontium or regulates dental follicle stem/progenitor cells is unknown.MethodsDental follicle stem/progenitor cells were isolated from postnatal day 1 (p1) to p11 from rat mandibular first molars. Immunolocalization mapped Wnt5a expression in the alveolar bone, periodontal ligament, and the developing ameloblast and odontoblast layers. Mononucleated and adherent cells were isolated from p7 dental follicle. Wnt5a was overexpressed in dental follicle stem/progenitor cells to study their proliferation, osteogenic differentiation and migration behavior, with subpopulations of native dental follicle stem/progenitor cells as controls, using real-time PCR (Taqman), Lenti-viral transfection, Western blotting and immunofluorescence.ResultsWnt5a was expressed consistently in p1 to p11 rat peridontium. Native, p7 dental follicle stem/progenitor cells had modest ability to mineralize in the tested 14 days. Even in chemically defined osteogenesis medium, dental follicle stem/progenitor cells only showed modest mineralization. Upon addition of 300 ng/mL Wnt5a protein in osteogenesis medium, dental follicle stem/progenitor cells displayed mineralization that was still unremarkable. Chemically induced or Wnt5a-induced mineralization of dental follicle cells only occurred sparsely. Combination of Wnt5a with 100 ng/mL BMP2 finally prompted dental follicle stem/progenitor cells to produce robust mineralization with elevated expression of Runx2, alkaline phosphatase, collagen 1α1 and osteocalcin. Thus, native dental follicle stem/progenitor cells or some of their fractions may be somewhat modest in mineralization. Strikingly, Wnt5a protein significantly augmented RANKL ligand, suggesting putative regulatory roles of dental follicle stem/progenitor cells for the monocyte/osteoclast lineage and potential involvement in alveolar bone remodeling and/or resorption. P-Jnk1/2 was activated in Wnt5a overexpressed dental follicle cells; conversely, exposure to SP600125, a c-Jun N-terminal kinase (JNK) inhibitor attenuated Runx2, collagen 1α1 and osteocalcin expression either in the presence or absence of Wnt5a. Wnt5a overexpression in dental follicle stem/progenitor cells significantly reduced their proliferation rates, but robustly augmented their migration capacity.ConclusionsThese findings provide a glimpse of Wnt5a’s putative roles in dental follicle stem/progenitor cells and the periodontium with implications in periodontal disease, tooth eruption, dental implant bone healing and orthodontic tooth movement.

Contribution of Donor and Host Mesenchyme to the Transplanted Tooth Germs

Autologous tooth germ transplantation of immature teeth is an alternative method of tooth replacement that could be used instead of dental implants in younger patients. However, it is paramount that the dental pulp remain vital and that root formation continue in the transplanted location. The goal of this study is to characterize the healing of allogenic tooth grafts in an animal model using GFP-labeled donor or host postnatal mice. In addition, the putative stem cells were labeled before transplantation with a pulse-chase paradigm. Transplanted molars formed cusps and roots and erupted into occlusion by 2 wk postoperatively. Host label-retaining cells (LRCs) were maintained in the center of pulp tissue associating with blood vessels. Dual labeling showed that a proportion of LRCs were incorporated into the odontoblast layer. Host cells, including putative dendritic cells and the endothelium, also immigrated into the pulp tissue but did not contribute to the odontoblast layer. Therefore, LRCs or putative mesenchymal stem cells are retained in the transplanted pulps. Hertwig’s epithelial root sheath remains vital, and epithelial LRCs are present in the donor cervical loops. Thus, the dynamic donor-host interaction occurred in the developing transplant, suggesting that these changes affect the characteristics of the dental pulp.

Histologic evaluation of apical pulp of immature apex following extraction, surface treatment, and replantation in different storage media in dogs.

This study was designed to evaluate the apical pulp of open apex teeth following extraction, surface treatment, and preservation in different storage media in dogs. Four healthy male dogs were included in this prospective animal study. In the first phase of extraction, 30 teeth were extracted atraumatically from dogs (incisors and premolars) and then randomly divided into three groups based on different types of storage media: (i) milk, (ii) HBSS, and (iii) dry storage. In milk and HBSS groups, teeth were contaminated by dog's saliva and then kept in milk and HBSS solution, respectively, for 2 h. In the third group, teeth were stored in a dry storage for 2 h. Before replantation, all teeth were soaked in 10% doxycycline. After 2 months, in the second phase of extraction, all experimental teeth were extracted (40 teeth including control group). Three longitudinal sections (3-4 mm thickness) were stained with H&E and investigated under optical microscope. Histologic evaluations were performed, and data were analyzed using Kruskal-Wallis and Fisher's exact test (α = 0.05). There was a significant difference between the milk and control groups regarding the presence of the odontoblastic layer and resorption (P = 0.04). Also, there was a significant difference between dry storage and control group in the presence of the odontoblastic layer (P = 0.02). There was no statistically significant difference in other histologic factors between different groups (P > 0.05). It was revealed that there is no significant difference between different groups regarding the intensity (P = 0.06) and the type of the inflammation (P = 0.24). Also, the hyperemia showed no significant difference between different groups (P = 0.51). It was concluded that HBSS and milk are appropriate storage media. Also, it was shown that 2 h dry storage probably does not threaten the vitality of the pulp cells.

Low-level laser therapy as an alternative for pulpotomy in human primary teeth

This study aimed to evaluate the effects of low-level laser therapy (LLLT) on pulpal response of primary teeth. Twenty mandibular primary molars were randomly divided into the following groups: group I Buckley's formocresol (diluted at 1:5), group II calcium hydroxide, group III LLLT + zinc oxide/eugenol, and group IV LLLT + calcium hydroxide. LLLT parameters were set at 660-nm wavelength, 10-mW power output, and 2.5 J/cm(2) energy density for 10 s in continuous mode (InGaAlP laser, Twin Laser®, MMOptics, Sao Carlos, Sao Paulo, Brazil). The teeth were extracted at the regular exfoliation period. The dentin-pulp complex was graded by an established histopathological score system. Statistical analysis was performed by Kruskal-Wallis and chi-square test. The histopathological assessment revealed statistically significant differences among groups (P < 0.05). The lowest degree of pulpal inflammation was present in LLLT + calcium hydroxide (P = 0.0296). Calcium hydroxide showed the highest rate of hard tissue barrier (P = 0.0033), odontoblastic layer (P = 0.0033), and dense collagen fibers (P = 0.0095). On the other hand, formocresol showed the highest incidence of internal resorption (P = 0.0142). Based on this study, low-level laser therapy preceding the use of calcium hydroxide exhibited satisfactory results on pulp tissue healing. However, further clinical studies on human teeth with long-term follow-up are needed to test the low-level laser therapy efficacy.

Abnormal osteopontin and matrix extracellular phosphoglycoprotein localization, and odontoblast differentiation, in X-linked hypophosphatemic teeth

Mutations in phosphate-regulating gene (PHEX) lead to X-linked hypophosphatemic rickets (XLH), a genetic disease characterized by impaired mineralization in bones and teeth. In human XLH tooth dentin, calcospherites that would normally merge as part of the mineralization process are separated by unmineralized interglobular spaces where fragments of matrix proteins accumulate. Here, we immunolocalized osteopontin (OPN) in human XLH teeth, in a three-dimensional XLH human dental pulp stem cell-collagen scaffold culture model and in a rat tooth injury repair model treated with acidic serine- and aspartate-rich motif peptides (ASARM). In parallel, matrix extracellular phosphoglycoprotein (MEPE) immunolocalization and alkaline phosphatase (ALP) activity were assessed in XLH teeth. OPN was expressed by odontoblasts in the XLH models, and localized to the abnormal calcospherites of XLH tooth dentin. In addition, ALP activity and MEPE localization were abnormal in human XLH teeth, with MEPE showing an accumulation in the unmineralized interglobular spaces in dentin. Furthermore, XLH odontoblasts failed to form a well-polarized odontoblast layer. These data suggest that both MEPE and OPN are involved in impaired tooth mineralization associated with XLH, possibly through different effects on the mineralization process.

Is hard tissue formation in the dental pulp after the death of the primary odontoblasts a regenerative or a reparative process?

ObjectivesConceptually, two types of tertiary dentine may be produced in response to caries and environmental irritations: “reactionary dentine” that is secreted by existing primary odontoblasts and “reparative dentine”, formed after the death of the odontoblasts by proliferation and differentiation of progenitor cells into odontoblast-like cells. Because histologic evidence for tubular dentine generated by newly differentiated odontoblast-like cells is lacking in human teeth, the present study examined pulpal cellular changes associated with caries/restorations, in the presence or absence of pulpal exposures. MethodsNinety-six extracted human teeth were histologically processed and serial sectioned for light microscopy: 65 contained untreated enamel/dentine caries; 20 were heavily restored and 11 had carious exposures managed by direct pulp-capping. ResultsSparsely distributed, irregularly arranged dentinal tubules were identified from the tertiary dentine formed in teeth with unexposed medium/deep caries and in restored teeth; those tubules were continuous with the tubules of secondary dentine; in some cases, tubules were absent. The palisade odontoblast layer was reduced to a single layer of flattened cells. In direct pulp-capping of pulp exposures, the defects were repaired by the deposition of an amorphous dystrophic calcified tissue that resembled pulp stones more than dentine, sometimes entrapping pulpal remnants. This atubular hard tissue was lined by fibroblasts and collagen fibrils. ConclusionsHistological evidence from the present study indicates that reparative dentinogenesis cannot be considered as a regenerative process since the so-formed hard tissue lacks tubular features characteristic of genuine dentine. Rather, this process represents a repair response that produces calcified scar tissues by pulpal fibroblasts. Clinical significanceFormation of hard tissue in the dental pulp after the death of the primary odontoblasts has often been regarded by clinicians as regeneration of dentine. If the objective of the clinical procedures involved is to induce healing, reduce dentine hypersensitivity, or minimise future bacteria exposure, such procedures may be regarded as clinical success. However, current clinical treatment procedures are not adept at regenerating physiological dentne because the tissues formed in the dental pulp are more likely the result of repair responses via the formation of calcified scar tissues.

Absent in melanoma 2 (AIM2) expressed in human dental pulp mediates IL-1β secretion in response to cytoplasmic DNA.

The inflammasome has been determined to play an important role in inflammatory diseases in recent years. Absent in melanoma 2 (AIM2), an inflammasome that recognizes cytoplasmic DNA, has recently been identified as a critical regulator of immune responses. In this study, we explored whether AIM2 was expressed in human dental pulp and defined the role of AIM2 in regulating interleukin (IL)-1β secretion. We demonstrated that AIM2 was only detected in the odontoblast layer of healthy dental pulp, whereas strong expression was observed in inflamed dental pulp. Stimulation with interferon gamma (IFN-γ) and cytoplasmic DNA significantly activated the AIM2 inflammasome and increased IL-1β secretion in human dental pulp cells (HDPCs) in a time- and dose-dependent manner. Moreover, the knockdown of AIM2 downregulated both cleaved-caspase-1 expression and IL-1β release in HDPCs. These results suggest that AIM2 expressed in human dental pulp plays an important role in the immune defense by activating the inflammasome signaling pathway.

Reactionary Dentinogenesis and Neuroimmune Response in Dental Caries

Reactionary dentin formation is an adaptive secretory response mediated by odontoblasts to moderate dentin injury. The implications of this process for neuroimmune interactions operating to contain pathogens have not been fully appreciated. The purpose of the present study was to describe the relationship between reactionary dentinogenesis, the neurogenic changes of dental pulp innervation, and dendritic cell recruitment to caries progression, using a comparative immunohistochemical approach in human teeth from young adult individuals. Reactionary dentin formation during dentin caries progression is associated with changes in the integrity of junctional complexes within the odontoblast layer. Diminished coexpression of Cx43 and zonula occludens 1 implies a reduced level of intercellular connectivity between odontoblasts. Dentin caries also causes overexpression of growth-associated protein 43, a modulator of neural plasticity that promotes extensive sprouting of nerve endings into the reactionary dentin matrix. At the same time, an elevated number of HLA-DR-positive dendritic cells infiltrate the odontoblast layer and subsequently invade reactionary dentin formed underneath the early caries-affected regions. Simultaneous odontoblast layer remodeling, nerve fiber sprouting, and activation of dendritic cells during caries progression suggest a coordinated neuroimmune response to fight caries pathogen invasion and to promote dentin-pulp healing. We propose that reactionary dentin formation hinders pathogen invasion and supports defensive neuroimmune interactions against infection. The eventual understanding of this complex scenario may contribute to the development of novel approaches to dental caries treatment.

Hypoxic condition promotes differentiation and mineralization of dental pulp cells in vivo

To investigate the behaviour of dental pulp cells under hypoxic conditions in vivo using an experimental animal model. A mini-screw was inserted into the inferior dental nerve canal of rats to arrest the blood supply, which resulted in a reduced oxygen level in the dental pulps of molar teeth used for the experimental group. The decrease in blood supply was evaluated by injected India ink in transparent specimens. The hypoxia marker hypoxyprobe-1 was investigated by immunohistochemical staining. The mRNA expressions of ATP-binding cassette transporter (ABC) G2 (ABCG2) which is a marker for the capacity to excrete metabolites and for stem-like cells as well as dentine sialophosphoprotein (DSPP) and osteocalcin (OCN) which are markers for mineralization were evaluated by RT-PCR. Protein was evaluated by immunohistochemical staining using ABCG2, dentine sialoprotein (DSP) and OCN. The evaluation of India ink indicated a decreased blood supply in the transparent specimens, and hypoxyprobe-1 immunohistochemical staining showed positive expression. ABCG2, DSPP and OCN mRNA expressions increased at 7 and 14 days. Immunohistochemically, ABCG2, DSP and OCN-positive cells were localized in the odontoblastic layer. Hypoxic conditions promoted mineralization and differentiation of dental pulp cells of the odontoblastic layer.

TLR4 Activation by Lipopolysaccharide and Streptococcus mutans Induces Differential Regulation of Proliferation and Migration in Human Dental Pulp Stem Cells

IntroductionDental pulp stem cells (DPSCs) are suspected to be an important part of the innate immune response of dental pulp, which is triggered by microorganisms that progressively invade the human tooth during the formation of caries. This study was performed to elucidate the expression of toll-like receptor 4 (TLR4) in dental pulp of deep caries and to determine whether TLR4 modulates the proliferation and migration of DPSCs. MethodsPulp tissue samples were collected from freshly extracted human wisdom tooth. Immunohistochemistry and immunofluorescence were performed to determine the distribution of TLR4 in healthy dental pulp and dental pulp in deep caries. DPSCs were cultured and purified by collecting multiple colonies. The proliferation and migration of DPSCs were examined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide, scratch test, and transwell migration assay after stimulation with lipopolysaccharide and extracts from Streptococcus mutans. TLR4 messenger RNA (mRNA) and cytokine mRNA were evaluated with real-time polymerase chain reaction; TLR4 protein was examined with Western blot and immunocytochemistry. ResultsTLR4 is expressed in the odontoblast layer and areas that colocalize with blood vessels to different levels in healthy teeth and teeth affected by caries. TLR4 mRNA, TLR4 protein, and mRNA of cytokine levels can be elevated with stimulations of LPS and extracts from S. mutans. Lipopolysaccharide and extracts from S. mutans treatment inhibited the proliferation of DPSCs but promoted migration; however, these changes were abolished when TLR4 was blocked by anti-TLR4 antibody. ConclusionsThese results suggest that TLR4 will be activated and regulate the proliferation and migration of DPSCs in deep caries. TLR4 may play an important role in the immune response by DPSCs.

Comparison of Pulpal Responses to Pulpotomy and Pulp Capping with Biodentine and Mineral Trioxide Aggregate in Dogs

IntroductionThis study evaluated the pulpal and periapical responses of dogs' teeth after pulpotomy and pulp capping with a new tricalcium silicate–based cement (Biodentine) when compared with mineral trioxide aggregate (MTA) by radiographic, histopathologic, and histomicrobiological analyses. MethodsSixty roots (30 teeth) of dogs were divided into 2 groups, Biodentine (n = 36 roots) and ProRoot MTA (control, n = 24 roots). Animals were killed after 120 days, and the teeth were subjected to histotechnical processing (hematoxylin-eosin and Brown and Brenn staining). Qualitative and quantitative histopathologic data were analyzed by Fisher exact and Mann-Whitney tests (α = 0.05). ResultsRadiographically, mineralized tissue bridge formation was observed in more specimens treated with Biodentine (96.8%) than with MTA (72.2%) (P = .02). Integrity of the lamina dura and absence of periapical bone rarefaction and root resorption (external and internal) were observed in all specimens. Histopathologic and histomicrobiological analyses revealed mineralized tissue bridge formation, pulpal vitality, odontoblast layer integrity, preserved periodontal ligament, and absence of bone or root resorption and microorganisms in both groups. Although the bridges formed at the amputation site had similar morphology, they were significantly thicker in the Biodentine group (P < .0001). Comparison between the radiographic and histopathologic results showed that radiographic visualization of more bridges in the Biodentine group was related to bridge thickness because radiographic diagnosis was flawed for bridges with thickness less than 0.5 mm. Fluorescence microscopy improved the visualization of bridge structure. ConclusionsBiodentine presented tissue compatibility and allowed for mineralized tissue bridge formation after pulpotomy in all specimens with similar morphology and integrity to those formed with use of MTA. Periapical radiographs failed in detecting mineralized tissue bridges with thickness less than 0.5 mm.

Transcriptional Factor ATF6 is Involved in Odontoblastic Differentiation

ATF6 is an endoplasmic reticulum (ER) membrane-bound transcription factor that regulates various cellular functions. The purpose of this study was to investigate the role of ATF6 in odontoblast differentiation. Rat tooth germs were isolated, changes in gene expression were evaluated over time, and localization of ATF6 was determined by immunohistochemistry. Human dental pulp cells (HDPCs) were cultured with 50 µg/mL ascorbic acid and 5 mmol/L β-glycerophosphate or 100 ng/mL bone morphogenetic protein 2 to induce differentiation. Translocation of ATF6 was observed by immunofluorescence and confocal microscopy. Overexpression of ATF6 was performed with an adenoviral vector. Matrix mineralization was evaluated by alizarin red staining. Immunoreactivity to anti-ATF6 was observed in the odontoblastic layer of the molar tooth germ, and expressions of ATF6, dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) increased gradually during tooth germ development. When HDPCs were cultured in differentiation media, ATF6, DSPP, and DMP1 expression increased with the expression of unfolded protein response (UPR) markers, BiP and CHOP. Immunofluorescence results showed that ATF6 protein moved from cytoplasm to nucleus when cells were exposed to differentiation media. Notably, overexpression of ATF6 increased DSPP and DMP1 expression, alkaline phosphatase (ALP) activity, and matrix mineralization in HDPC cultures. Inhibition of ATF6 decreased ALP activity and mineralization. These results suggest that ER membrane-bound transcriptional factor ATF6 may be involved in odontoblastic differentiation.