Rat Molar Tooth Research Articles

Histological evaluation of different concentrations of hyaluronic-acid-added zinc oxide eugenol on rat molar pulp.

Hyaluronic acid (HA), known for diverse properties, was investigated for its potential in dental pulp therapy. This study investigated the potential of HA in dental pulp therapy by examining the physical properties and effects of zinc oxide eugenol (ZOE) pulpotomy materials containing varying HA concentrations on rat molar teeth. In vitro tests assessed compressive strength and hardness of ZOE materials blended with HA (0.5%, 1%, 3%) and HA gels (0.54%, 0.8%). 120 samples, encompassing the control group, underwent compressive strength testing, while 60 samples were designated for hardness assessment. In vivo experiments on rat molars studied histological effects of HA-containing ZOE on dental pulp over 1week and 1month. Gels with HA concentrations of 0.5%, 1%, and 0.54% were used in pulpotomy on 22 rats. Each rat underwent the procedure on four teeth, with one tooth serving as a control, totaling 88 teeth subjected to the intervention. In the analyses, SPSS 22.0 was used and the significance level was set at P = 0.05. Findings showed that HA at 0.5% maintained compressive strength, but higher concentrations decreased mechanical properties significantly (P = 0.001). Histological assessments indicated better outcomes with lower HA concentrations in terms of odontoblast layer continuity (P = 0.005 at 1month) and pulp vitality (P = 0.001 at 1week and P = 0.018 at 1month). The study suggests HA holds promise for pulpotomy and regenerative endodontic treatments, but further research is needed to understand long-term clinical implications.

Cavity preparation model in rat maxillary first molars: A pilot study

Objective: To conduct a standardized method for cavity preparation on the palatal surface of rat maxillary molars and to introduce a standardized method for tooth correct alignment within the specimen during the wax embedding procedure to better detect cavity position within the examined slides. Materials and methods: Six male Wistar rats, aged 4-6 weeks, were used. The maxillary molars of three animals were sectioned in the frontal plane to identify the thickness of hard tissue on the palatal surface of the first molar which was (250-300µm). The end-cutting bur (with a cutting head diameter of 0.2mm) was suitable for preparing a dentinal cavity (70-80µm) depth. Cavity preparation was then performed using the same bur on the tooth surface in the other three animals. Rats are then euthanized before dissecting, fixing, and demineralizing the teeth. For better alignment of teeth samples during the waxing procedure, K-file endodontic instrument size #8 was dipped in Indian ink. The file tip was inserted on the jaw bone at the buccal side of the tooth in a region opposed to the prepared cavity on the palatal side. Moreover, a small Dycal applicator instrument was used to mark the jaw bone on the mesial side of teeth samples as an orientation for the cutting surface. Results: Well-defined sections were obtained with a clear cavity extension within dentin and without any signs of pulp exposure in all samples. Conclusion: This pilot was conducted to perform an easy procedure for cavity preparation in rat molar teeth to obtain a clear histopathological section.

Experimental study of dexamethasone-loaded hollow hydroxyapatite microspheres applied to direct pulp capping of rat molars.

Dexamethasone (DEX) exerts anti-inflammatory and osteogenic effects. Hydroxyapatite is commonly used in bone repair due to its osteoconductivity, osseointegration, and osteogenesis induction. Hollow hydroxyapatite (HHAM) is often used as a drug carrier. This study aimed to investigate the histological responses of exposed dental pulp when dexamethasone-loaded nanohydroxyapatite microspheres (DHHAM) were used as a direct capping agent. Cavities were created in the left maxillary first molar of Wistar rats and filled with Dycal, HHAM, and DHHAM. No drug was administered to the control group. The rats were sacrificed at 1, 2, and 4 weeks after the procedure. The molars were extracted for fixation, demineralization, dehydration, embedding, and sectioning. H&E staining was performed to detect the formation of reparative dentin. H&E and CD45 immunohistochemical staining were performed to detect pulp inflammation. Immunohistochemical staining was performed to assess the expressions of dentin matrix protein 1 (DMP-1), interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β. The results of H&E and CD45 immunohistochemical staining showed that the degree of inflammation in the DHHAM group was less than that in the Control and HHAM groups at 1, 2, and 4 weeks after capping of the rat molar teeth (p<0.01). The H&E staining showed that the percentage of reparative dentin formed in the DHHAM group was higher than that in the Control, HHAM (p<0.001), and Dycal groups (p<0.01) at 1 and 2 weeks, and was significantly higher than that in the Control group (p<0.001) and the HHAM group (p<0.01) at 4 weeks. The immunohistochemical staining showed a lower range and intensity of expression of IL-1β, IL-6, and TNF-α and high expression levels of DMP-1 in the DHHAM group at 1, 2, and 4 weeks after pulp capping relative to the Control group. DHHAM significantly inhibited the progression of inflammation and promoted reparative dentin formation.

A New Experimental Technique for Complete Extraction of Mandibular First Molar Teeth in Rats.

Intact extraction of the mandibular first molar tooth is an interesting model for studies of alveolar bone healing. The aim of this study was to describe a new experimental technique for extraction of rat mandibular first molar teeth with crown and all 4 roots intact using controlled forces applied to the teeth. One hundred and twenty female Sprague-Dawley rats were used from a center for experimental animal research. Animals underwent general anesthesia and were then placed in a special dental unit (designed by Moghadam) for the extraction of rat teeth. After syndesmotomy, luxation of the tooth began with a tipping movement in the buccal direction with a very low range of motion for 1 s. A tipping movement in the lingual direction was then used to continue luxation. After a maximum of 10 repetitions, the tooth was left alone for 30 s. After 3-4 stages of this cycle, the tooth loosened. To complete the luxation, the same forces were applied in the buccal and lingual directions with larger amplitude for 3 s. After this step, the tooth was loose enough to be easily extracted. The alveolus was then sutured closed. The results showed no hemorrhage or fracture of crowns and mesial or distal roots, and only 8% of the buccal and lingual roots fractured. The technique designed and used in this study was shown to be an effective model for complete molar tooth extraction in the rat. This technique could also be used in the treatment of other rodents.

Encapsulation of human endometrial stem cells in chitosan hydrogel containing titanium oxide nanoparticles for dental pulp repair and tissue regeneration in male Wistar rats

This study aimed to determine the impact of human endometrial stem cells (EnSCs) and titanium oxide nanoparticles (TiO2 NPs) on dental pulp repair and regeneration in an animal model through dentine development and tissue regeneration. The EnSCs were put on a three-dimensional (3D) chitosan scaffold containing TiO2 NPs after obtaining and purifying the collagenase enzyme. Pulps were exposed on the maxillary left first molar of all rats followed by direct pulp capping with the experimental scaffolds, as follows. Groups were: 1, control group without any treatment; 2, chitosan group (CS); 3, chitosan group with stem cells (CS/SCs); 4, chitosan group with stem cells and TiO2 NPs (CS/EnSCs/TiO2). Glass ionomer was used as a sealant in all groups. The teeth were extracted and histologically evaluated after 8 weeks. The quality and amount of dentine in the CS/EnSCs/TiO2 group were higher than in the other groups. The combination of EnSCs with TiO2 NPs and 3D chitosan scaffolds had a synergistic effect on each other, evidencing increased speed and quality of dentine formation. Using EnSCs with TiO2 NPs on a 3D chitosan scaffold can be a suitable combination for direct pulp capping and dentine regeneration in a rat molar tooth model.

Immunohistochemical localization of CD146 and alpha-smooth muscle actin during dentin formation and regeneration.

Cluster of differentiation 146 (CD146) is known to localize in stem cells and precursor cells of various tissues. In this study, to analyze the function of CD146 in odontoblast differentiation, immunohistochemical localization of CD146 was examined during rat molar tooth development and after cavity preparation. At the cap and bell stages, many CD146-positive cells were visible around the blood vessels in the dental papillae. On Postnatal day 2, osterix-positive odontoblasts were arranged in the dentin sialoprotein (DSP)-positive predentin, and many CD146-positive cells were observed near these odontoblasts with blood vessels. Some perivascular CD146-positive cells overlapped with Smad4-positive cells. However, the immunoreactivity for alpha-smooth muscle actin (α-SMA), one of the markers for undifferentiated cells, was negligible. Furthermore, the number of these cells decreased in the dental pulp on Postnatal day 28. On Day 4 after cavity preparation, Osterix-positive odontoblasts appeared lining the reparative dentin. Most of the blood vessels near the reparative dentin showed immunoreactivities for CD146. Reparative odontoblasts actively formed DSP-positive dentin matrix because these cells were positive for Smad4 and Osterix, but not for α-SMA. After 7 days, the number of CD146-positive cells near blood vessels decreased in the dental pulp beneath the cavity. These results suggest that the CD146 is expressed in the perivascular area of the dental pulp and induces vascularization in the vicinity of dentin formation, and some CD146-positive cells are activated by the bone morphogenetic protein signaling pathway and differentiate into odontoblasts in the early stages of dentin formation and repair.

Evaluation of the transdentinal capability of the intrinsic antibacterial cetylpyridinium chloride/cholesterol sterosomes in vitro and in vivo.

Dentinal tubules serve as disease-causing channels for infiltration and penetration of bacteria and their by-products; which are regarded as the major driver of pathogenesis in pulpal inflammation and infection. In this study, we aimed to evaluate the transdentinal potential of nanoscale cetylpyridinium chloride/cholesterol (CPC/Chol) sterosomes, which are a recently developed type of cationic non-phospholipid liposomal nanocarrier; as well as their intrinsic and universal antibacterial activity. Cetylpyridinium chloride/cholesterol sterosomes were formulated, with a hydrodynamic diameter of 134 ± 4nm, a low polydisperse index of 0.161 ± 0.007, and a positive zeta potential of 41 ± 3mV at pH7.4. Transdentinal diffusion ability of sterosomes was evaluated using human dentine blocks in vitro, and Wistar rat molar teeth in vivo. The intrinsic antibacterial activities of CPC/Chol sterosomes against Enterococcus faecalis, Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis were further examined. Cetylpyridinium chloride/cholesterol sterosomes successfully penetrated through the dentinal tubules, and diffused into the pulp, which could be internalized by dental pulp cells with a high efficiency. In addition, they exhibited substantial levels of intrinsic antibacterial activity against these Gram-positive and Gram-negative endodontic bacteria and their biofilms. Given its high penetration and diffusion ability through the dentine and pulp, great potential for multi-drug delivery, and distinct intrinsic antibacterial activity; sterosome-based nanocarriers might serve as a promising therapeutic strategy aimed at targeting various specific pathways associated with pulpal diseases. This will help determine and characterize the most appropriate prophylactic and therapeutic targets for early intervention in our future dentistry practice.

Pulpal Response to the Combined Use of Mineral Trioxide Aggregate and Iloprost for Direct Pulp Capping

Purpose: The present study aims to assess the combined effects of mineral trioxide aggregate (MTA) and iloprost when used as a pulp capping material on pulpal inflammation and tertiary dentin formation compared with MTA and iloprost alone in rat molar teeth. Methods: Eighty maxillary first molar rat teeth were exposed and capped with iloprost solution, MTA, or MTA mixed with iloprost (MTA-iloprost). The cavities were then filled with resin-modified glass ionomer. The cavity was restored with glass ionomer without the use of pulp capping agent in the control group. The rats were sacrificed after one and four weeks. Block sections of the molar specimens were prepared and subjected to hematoxylin and eosin staining for evaluation. Statistical analysis was done using the Kruskal–Wallis test, followed by Dunnett’s test. Results: At week one, the control group showed significantly more severe pulpal inflammatory reactions than the iloprost (p = 0.00), MTA (p = 0.04), and MTA-iloprost (p = 0.00) groups. Hard tissue formation was commonly found in the iloprost, MTA, and MTA-iloprost groups. After four weeks, pulpal tissue degeneration was observed in the control group. Complete hard tissue barriers were found in 50%, 72.7%, and 77.8% of the specimens in iloprost, MTA, and MTA-iloprost groups, respectively, with no significant differences among the experimental groups. The dentinal tubule patterns were mostly regular in the MTA-iloprost group and irregular in the iloprost and MTA groups. Conclusions: The application of iloprost, MTA, and MTA-iloprost as a pulp capping material resulted in similar pulpal responses in the mechanically exposed pulp of rat molars. Therefore, mixing MTA with iloprost might not be clinically significant.

Nanotherapeutics for regeneration of degenerated tissue infected by bacteria through the multiple delivery of bioactive ions and growth factor with antibacterial/angiogenic and osteogenic/odontogenic capacity

Therapeutic options are quite limited in clinics for the successful repair of infected/degenerated tissues. Although the prevalent treatment is the complete removal of the whole infected tissue, this leads to a loss of tissue function and serious complications. Herein the dental pulp infection, as one of the most common dental problems, was selected as a clinically relevant case to regenerate using a multifunctional nanotherapeutic approach. For this, a mesoporous bioactive glass nano-delivery system incorporating silicate, calcium, and copper as well as loading epidermal growth factor (EGF) was designed to provide antibacterial/pro-angiogenic and osteo/odontogenic multiple therapeutic effects. Amine-functionalized Cu-doped bioactive glass nanospheres (Cu-BGn) were prepared to be 50–60 nm in size, mesoporous, positive-charged and bone-bioactive. The Cu-BGn could release bioactive ions (copper, calcium and silicate ions) with therapeutically-effective doses. The Cu-BGn treatment to human umbilical vein endothelial cells (HUVEC) led to significant enhancement of the migration, tubule formation and expression of angiogenic gene (e.g. vascular endothelial growth factor, VEGF). Furthermore, the EGF-loaded Cu-BGn (EGF@Cu-BGn) showed pro-angiogenic effects with antibacterial activity against E. faecalis, a pathogen commonly involved in the pulp infection. Of note, under the co-culture condition of HUVEC with E. faecalis, the secretion of VEGF was up-regulated. In addition, the osteo/odontogenic stimulation of the EGF@Cu-BGn was evidenced with human dental pulp stem cells. The local administration of the EGF@Cu-BGn in a rat molar tooth defect infected with E. faecalis revealed significant in vivo regenerative capacity, highlighting the nanotherapeutic uses of the multifunctional nanoparticles for regenerating infected/damaged hard tissues.

Influence of low-level laser therapy on inflammation, collagen fiber maturation, and tertiary dentin deposition in the pulp of bleached teeth.

We evaluated the effects of low-level laser therapy (LLLT) using an infrared laser (IRL) and a red laser (RL) on the pulp of molar teeth in rats after dental bleaching to assess inflammation, collagen fiber maturation, and tertiary dentin formation. Eighty Wistar rats (Rattus norvegicus, albinus) were randomly divided into eight groups with 10 hemimaxillae in each of the following: control; bleached (Ble, 35% hydrogen peroxide [H2O2]); Ble-1IRL and Ble-1RL (one IRL [808nm, 30s, 3J] or RL [660nm, 15s, 1.5J] application immediately after H2O2); Ble-3IRL and Ble-3RL (three [immediately, 24h, and 48h] IRL or RL applications after H2O2); and 3IRL and 3RL (three IRL or RL applications without bleaching). The rats were euthanized after 2 and 30days for histological evaluation of inflammation (hematoxylin-eosin) and maturation of collagen fibers (picrosirius red). Additionally, the dentin deposition in the specimens obtained at 30days was quantified via microtomography of the pulp chamber volume. Statistical analyses were performed (P < 0.05). Initially, severe damages to the pulp were observed in the Ble and Ble-1RL groups. Ble-1IRL and Ble-3RL groups showed lower inflammation. The bleached groups had a greater amount of mature collagen fibers than the control group. The Ble-3IRL group had a greater number of immature fibers than the Ble group. At 30days, there was an absence of inflammation and equal proportion of mature and immature collagen fibers. All bleached groups showed a reduction in the volume of the pulp chamber. Three consecutive applications of RL and one IRL application can minimize damage to the pulp of bleached teeth, whereas three IRL applications can minimize pulp fibrosis. However, LLLT did not prevent deposition of tertiary dentin. This study describes LLLT protocols capable of minimizing inflammation and maturation of collagen fibers in pulp tissue after dental bleaching. However, the protocols proved insufficient for reducing the formation of tertiary dentin in bleached teeth.

Notch Coordinates Periodontal Ligament Maturation through Regulating Lamin A

Tooth eruption is a continuous biological process with dynamic changes at cellular and tissue levels, particularly within the periodontal ligament (PDL). Occlusion completion is a significant physiological landmark of dentition establishment. However, the importance of the involvement of molecular networks engaging in occlusion establishment on the final PDL maturation is still largely unknown. In this study, using rat and mouse molar teeth and a human PDL cell line for RNAseq and proteomic analysis, we systematically screened the key molecular links in regulating PDL maturation before and after occlusion establishment. We discovered Notch, a key molecular pathway in regulating stem cell fate and differentiation, is a major player in the event. Intercepting the Notch pathway by deleting its key canonical transcriptional factor, RBP-Jkappa, using a conditional knockout strategy in the mice delayed PDL maturation. We also identified that Lamin A, a cell nuclear lamina member, is a unique marker of PDL maturation, and its expression is under the control of Notch signaling. Our study therefore provides a deep insight of how PDL maturation is regulated at the molecular level, and we expect the outcomes to be applied for a better understanding of the molecular regulation networks in physiological conditions such as tooth eruption and movement and also for periodontal diseases.

Chewing causes rapid changes in immunoreactive nerve patterns in rat molar teeth: Implications for dental proprioception and pain

ObjectiveThis study tests the hypothesis that normal use of teeth (chewing) causes changes in immunoreactive-(IR) patterns for endings of large Aβ and CGRP axons in rat molar cusps. DesignFirst, a new paradigm to test chewing in adult male rats was developed. Then IR patterns for large dental axons were analysed for a calcium-binding protein, parvalbumin (PV), heavy neurofilament protein-200 (NFP), and vesicle-release molecule synaptophysin (SYN) that all typify large dental axons and proprioceptors for comparison with endings of CGRP-IR neuropeptide axons. The behavior groups were: (1) daytime sleeping/fasting (Group:SF); (2) brief feeding after 8–11 h of daytime sleeping/fasting (Group:SF–C); (3) normal nocturnal feeding (Group:N); (4) nocturnal fasting (Group:NF); (5) brief feeding/chewing after nocturnal fasting (Group:NF–C). ResultsNerve endings with NFP-, PV-, or SYN-IR were lost or altered in pulp and dentin in all chewing groups. Other endings with CGRP-IR were near those with PV-, NFP- and SYN-IR at the pulp-dentin border and in dentin, and they also lost immunoreactivity in all chewing groups. The special beaded regions along the crown pulp/dentin borders lost neural labeling in all chewing groups. Nerves of molar roots and periodontal ligament were not changed. ConclusionsRapid neural reactions to chewing show extensive, reversible, non-nociceptive depletions of crown innervation. Those changes were rapid enough to occur during normal feeding followed by recovery during rest. The new dental paradigm related to chewing and fasting allows dissection of intradental proprioceptive-like mechanisms during normal tooth functions for comparison with nociceptive and mechanosensitive reactions after injury or inflammation.

Effects of Photobiomodulation and Ultrasound Applications on Orthodontically Induced Inflammatory Root Resorption; Transcriptional Alterations in OPG, RANKL, Cox-2: An Experimental Study in Rats

Objective: The aim of this study was to evaluate and compare the reparative and inhibitory effects of a light-emitting diode-mediated photobiomodulation (PBM) and of a low-intensity pulsed ultrasound (LIPUS) on orthodontically induced inflammatory root resorption (OIIRR). Materials and methods: Forty-nine Wistar rats were divided into four groups: untreated group (negative control), group treated with orthodontic appliances (positive control), PBM-treated group (wavelength: 618 nm, output power density: 20 mW/cm2), and LIPUS-treated group (frequency: 1.5% ± 5% MHz, pulse repetition ratio: 1.0% ± 10% kHz, effect area: 3.88% ± 1% cm2 and intensity: 30% ± 30% mW/cm2). OIIRR was induced experimentally in rats for 14 days with an applied force of 100g, and therapeutic approaches were performed concurrently. At the end of the experiment, upper first molar teeth of rats were prepared for genetic analysis, scanning electron microscopy, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining. Kruskal-Wallis and post hoc Dunn's tests were performed. Results: Number of osteoclasts (p < 0.01), number of resorption lacunae and resorption area ratio (p < 0.001) decreased and number of total cells (p < 0.001) increased with the PBM and LIPUS applications when compared with the positive control group. Receptor activator of nuclear factor kappa B ligand (RANKL) levels of PBM and LIPUS groups were lower (p < 0.001), and osteoprotegerin (OPG) levels were higher (p < 0.001) than the positive control group. Cyclooxygenase-2 (Cox-2) expression significantly decreased with LIPUS and PBM administrations (p < 0.05). No significant difference was observed among PBM and LIPUS groups. Conclusions: PBM and LIPUS applications showed marked inhibitory and reparative effects on OIIRR by modulating the OPG/RANKL ratio, Cox-2 expression, and cell differentiation of osteoblasts and osteoclasts.

A minimally invasive transfer method of mesenchymal stem cells to the intact periodontal ligament of rat teeth: a preliminary study.

The aim of this study was to introduce a minimally invasive procedure for mesenchymal stem cell (MSC) transfer into the intact periodontal ligament (PDL) of the molar teeth in rats. Ten 12-week-old Wistar albino rats were used for this preliminary study. MSCs were obtained from bones of two animals and were labeled with green fluorescent protein (GFP). Four animals were randomly selected for MSC injection, while 4 animals served as a control group. Samples were prepared for histological analysis, Cox-2 mRNA expression polymerase chain reaction analysis, and fluorescent microscopy evaluation. The number of total cells, number of osteoclastic cells, and Cox-2 mRNA expression levels of the periodontal tissue of teeth were calculated. The number of total cells was increased with MSC injections in PDL significantly (P < 0.001). The number of osteoclastic cells and Cox-2 mRNA expression were found to be similar for the two groups. GFP-labeled MSCs were observed with an expected luminescence on the smear samples of the PDL with transferred MSCs. The results of this preliminary study demonstrate successful evidence of transferring MSCs to intact PDL in a nonsurgical way and offer a minimally invasive procedure for transfer of MSCs to periodontal tissues.

Teneurin-2 presence in rat and human odontoblasts.

Teneurins are transmembrane proteins consisting of four paralogues (Ten-1-4), notably expressed in the central nervous system during development. All teneurins contain a bioactive peptide in their carboxyl terminal named teneurin C-terminal associated peptide (TCAP). The present study analyzed the detailed distribution of teneurin-2-like immunoreactive (Ten-2-LI) cells in developing and mature rat molar teeth, as well as in mature human dental pulps. Ten-2 and TCAP-2 genic expressions were also evaluated in rat and human dental pulps. Finally, Ten-2-LI cells were analyzed during the repair process after dentin-pulp complex injury in rat lower molar teeth. For this, histological sections of rat molar teeth and human dental pulps were submitted to immunohistochemical techniques, while total RNA from developing rat teeth and mature human dental pulps were submitted to conventional RT-PCR. Ten-2-LI cells were evident in the initial bell stage of rat molar teeth development, especially in ectomesenchymal cells of the dental papilla. Ten-2-LI odontoblasts showed strong immunoreactivity in rat and human mature teeth. Ten-2 and TCAP-2 genic expressions were confirmed in rat and human dental pulps. Dentin-pulp complex injury resulted in a decrease of Ten-2-LI odontoblasts after traumatic injury. Interestingly, Ten-2-LI cells were also evident in the pulp cell-rich zone in all postoperative days. In conclusion, Ten-2-LI presence in rat and human odontoblasts was demonstrated for the first time and Ten-2/TCAP-2 genic expressions were confirmed in rat and human dental pulps. Furthermore, it was revealed that Ten-2-LI rat odontoblasts can be modulated during the regenerative process.

Effects of tooth storage media on periodontal ligament preservation.

An easily available tooth storage medium is required to preserve a tooth after avulsion. Milk and Hank's balanced salt solution (HBSS) are recommended as tooth storage media, and egg white is also reported to be comparable with milk. The aim of this study was to histologically and immunohistochemically evaluate the effect of different tooth storage media on the periodontal ligament (PDL) of extracted teeth. This experiment used HBSS, milk, and egg white as tooth storage media. A total of ninety-six 6-week-old male Sprague-Dawley rats were used in these experiments. In each experiment, six rats were used for each medium and for the control group. Extracted rat molar teeth were immersed in these three different storage media for 1hour. In each medium, six samples (n=18) were fixed immediately, and the remaining samples (n=54) were subcutaneously transplanted. In the control group (n=24), the extracted teeth were fixed or transplanted immediately after extraction. At day 4, 1 and 2weeks after transplantation, the teeth were examined by radiographic, histological, and immunohistochemical methods. The number of PDL cells in the storage media was also counted. Teeth immersed for 1hour in milk showed the thinnest PDL. Immunohistochemistry of periostin and CD68 labeling suggested degradation of the extracellular matrix in the PDL. In the media used for immersion, more PDL cells were observed in milk than in the other solutions. After transplantation, the HBSS and egg white groups maintained adequate thickness of PDL but in the milk group, thinner PDL and ankylosis were observed. Adequate thickness of PDL was maintained in the egg white group, whereas the milk group showed disturbance in the PDL, which may lead to ankylosis.

Differential expression between “DSP-only” and DSP-PP523 transcripts in rat molar teeth

ObjectiveTo compare the expression patterns of two multiple transcripts derived from DSP-PP gene during tooth development. One is DSP-only transcript (i.e. does not encode PP) and the other is DSP-PP523 transcript, a main DSP-PP transcript. DesignUnique antisense and sense riboprobes were generated from DSP-only and DSPPP523 cDNAs for in situ studies to examine DSP-only and DSP-PP523 transcript expression in developing molars. Paraffin-embedded sections (5–7μ m) from embryonic 20day, postnatal 2, 3 and 6days were deparaffined and hydrated. Tissues were prehybridized, then hybridized with DSP-only and DSP-PP523 anti-sense (AS) or sense (S) Digoxigenin labeled-riboprobes overnight, and washed. Anti-Digoxigenin antibodies conjugated to alkaline phosphatase were used to detect the presence of bound riboprobes by color reaction with NBT/BCIP. Stro-1 antibody was used for immunohistochemical analysis of Stro-1 protein expression in rat molars. ResultsWe found that unlike the DSP-PP523 transcript, the DSP-only transcript does not express in the entire polarized mature odontoblasts but is expressed in the areas subjacent to the mature odontoblast layer. In addition, DSP-only transcript is expressed in the dental pulp. Interestingly, Stro-1 protein, a stem cell marker, was also identified in the areas subjacentto odontoblasts and in dental pulp. ConclusionDifferential expression of DSP-only and DSP-PP523 transcripts suggest that these two kinds of transcripts may play different roles during dentinogenesis. DSP-PP523 transcript is expressed in mature odontoblasts, which actively participates in dentin formation. DSP-only transcript might have a different function.

Immunohistochemical localization of SNARE core proteins in intrapulpal and intradentinal nerve fibers of rat molar teeth

ObjectiveThe present study was designed to elucidate whether three soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) core proteins, syntaxin-1, synaptosomal-associated protein of 25kDa (SNAP-25), and vesicle-associated membrane protein-2 (VAMP-2), are present in the dental pulp of the rat molar at both the light and electron microscopic levels. DesignImmunohistochemistry for protein gene product 9.5 (PGP 9.5), a pan-neuronal marker, syntaxin-1, SNAP-25, and VAMP-2 was performed on decalcified rat molars for light and electron microscopic analyses. Double-immunolabeling of PGP 9.5 and the SNARE core proteins, as well as combinations of the SNARE core proteins, was also carried out. ResultsPGP 9.5-immunoreactive nerve fibers ran toward the coronal region, ramified at the subodontoblast layer, and formed the subodontoblastic nerve plexus. Most nerve fibers penetrated the predentin and dentin along the dentinal tubules. Most, if not all, nerve fibers displayed immunoreactivity for syntaxin-1, SNAP-25, and VAMP-2. Immunoelectron microscopic analyses confirmed the presence of immunoreactivity for the SNARE core proteins within the intradental axonal elements. ConclusionsThe present findings suggest that, since SNARE core proteins participate in the docking and exocytosis of synaptic vesicles in the central nervous system, they may contribute to vesicle exocytosis from the dental nerve fibers even though there are no apparent synapses.

Evaluation of the Viability of Rat Periodontal Ligament Cells after Storing at 0℃/2 MPa Condition up to One Week: In Vivo MTT Method

Purpose: The aim of this study was to evaluate the rat periodontal ligament cell viability under 0℃/2 MPa condition up to one week using in vivo 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. Materials and Methods: As soon as 110 upper molar teeth of rats were extracted, they were stored in Hartman’s solution under 0℃/2 MPa condition for 1, 2, 3, 4, and 7 days each. All specimens were treated with in vivo MTT assay and the value of optical density was measured by ELISA reader. These values were statistically analyzed by one-way ANOVA. Result: There was no statistical difference on MTT value between immediate and 1 day storage group. There were statistically significant differences between 1 day and 2 days storage, 2 and 3 days storage groups, respectively. Teeth of 3, 4, and 7 days storage groups showed significantly lower MTT values compared with shorter period storage groups. Conclusion: When the MTT values were substituted in standard curve, 1 day storage group at 0℃/2 MPa condition showed 68% cell viability when compared with immediate group. It dropped to 13% at 2 days, and to less than 5% at 3 days or more.

Decreased face primary motor cortex (face-M1) excitability induced by noxious stimulation of the rat molar tooth pulp is dependent on the functional integrity of medullary astrocytes.

We have recently shown that application of the small-fiber excitant and inflammatory irritant mustard oil (MO) to the rat molar tooth pulp can decrease face-M1 excitability, but increase the excitability of trigeminal medullary dorsal horn (MDH) nociceptive neurons and that application of the astrocytic inhibitor methionine sulfoximine (MSO) to the face-M1 or MDH can attenuate the MO-induced changes. The present study aimed to determine whether medullary MSO application could modulate the MO-induced decreased face-M1 excitability. Under ketamine general anesthesia, electromyographic (EMG) electrodes were implanted into the right anterior digastric (RAD, jaw-opening muscle) of adult male Sprague-Dawley rats. A microelectrode was positioned at a low-threshold (≤30 μA) site in the left face-M1. Then MO (n = 16) or control-solution (n = 16) was applied to the previously exposed molar tooth pulp, and intracortical microstimulation threshold intensities for evoking RAD EMG activities were monitored for 15 min. MSO (0.1 mM, n = 8) or phosphate-buffered saline (PBS, n = 8) was then applied to the MDH and RAD thresholds monitored every 15 min for 120 min. Statistics used ANOVA followed by post hoc Bonferroni as appropriate (p < 0.05). As compared to baseline, RAD thresholds significantly increased (i.e., decreased excitability) within 1 min (26.3 ± 7.9%, p = 0.007) and peaked at 15 min following pulpal MO application (49.9 ± 5.7%, p < 0.001) but not following control-solution. Following MSO (but not PBS) application to the medulla, RAD thresholds significantly decreased within 15 min (26.5 ± 3.0%, p = 0.05) and at 60 min approached 6.3 ± 2.4%, of baseline values (p = 0.1). These novel findings suggest that clinically related motor disturbances arising from dental pain may involve decreased face-M1 excitability that is modulated by medullary astrocytes.