Periostin plays role in force-induced stem cell potential by periodontal ligament stem cells.

Mechanical stimuli have been shown to play an important role in directing stem cell fate and maintenance of tissue homeostasis. One of the functions of the mechanoresponsive tissue periodontal ligament (PDL) is to withstand the functional forces within the oral cavity. Periodontal ligament stem cells (PDLSCs) derived from periodontal tissue have been demonstrated to be able to respond directly to mechanical forces. However, the mechanisms of action of mechanical force on PDLSCs are not totally understood. The aim of this study was to investigate the mechanisms by which compressive force affects PDLSCs, especially their stemness properties. PDLSCs were established from extracted human third molars; their stem cell characteristics were validated by detecting the expression of stem cell markers and confirming their ability to differentiate into osteogenic and adipogenic lineages. PDLSCs were subjected to various magnitudes of static compressive force (0 [control], 0.5, 1.0, 1.5, or 2 g/cm2 ). Application of 1.0 g/cm2 compressive force significantly upregulated a panel of stem cell marker genes, including NANOG and OCT4. Conversely, higher force magnitudes downregulated these genes. Mechanical loading also upregulated periostin, a matrix protein that plays important roles in tissue morphogenesis. Interestingly, knockdown of periostin using siRNA abolished force-induced stem cell marker expression in PDLSCs. This study suggests a proper magnitude of compressive force could be one important factor involved in the modulation of the pluripotency of PDLSCs through the action of periostin. The precise mechanism by which periostin regulates stemness requires further detailed investigation.

TP53 polymorphisms in smokers' and nonsmokers' pericoronal follicles of asymptomatic impacted third molars.

BACKGROUND AND PURPOSE: The investigators designed and implemented a prospective cohort study composed of smoking and nonsmoking patients with asymptomatic fully impacted mandibular third molars. The objective of the paper was to evaluate 21 single-nucleotide polymorphisms (SNP) on the TP53 gene in smokers' (S) and nonsmokers' (NS) pericoronal follicles of asymptomatic impacted third molars. MATERIALS AND METHODS: Matrix-assisted desorption/ionization time of the flight mass spectrometry was used for SNP analysis of 21 regions in the TP53 gene. Descriptive statistics and Chi-square tests were computed with a P value of 0.05. RESULTS: : Ten of the 21 SNPs related to oral pathologies according to NCBI dbSNP, were detected; in these, the genotypic frequencies showed no differences between the S and NS groups (P > 0.05). The results showed a high ratio of SNPs without correlation between smoking and TP53 gene status. CONCLUSION: Further studies should examine the entire TP53 gene to elucidate how smoking affects it in larger study populations.

Dental pulp pluripotent-like stem cells (DPPSC), a new stem cell population with chromosomal stability and osteogenic capacity for biomaterials evaluation.

BACKGROUND: Biomaterials are widely used to regenerate or substitute bone tissue. In order to evaluate their potential use for clinical applications, these need to be tested and evaluated in vitro with cell culture models. Frequently, immortalized osteoblastic cell lines are used in these studies. However, their uncontrolled proliferation rate, phenotypic changes or aberrations in mitotic processes limits their use in long-term investigations. Recently, we described a new pluripotent-like subpopulation of dental pulp stem cells derived from the third molars (DPPSC) that shows genetic stability and shares some pluripotent characteristics with embryonic stem cells. In this study we aim to describe the use of DPPSC to test biomaterials, since we believe that the biomaterial cues will be more critical in order to enhance the differentiation of pluripotent stem cells. METHODS: The capacity of DPPSC to differentiate into osteogenic lineage was compared with human sarcoma osteogenic cell line (SAOS-2). Collagen and titanium were used to assess the cell behavior in commonly used biomaterials. The analyses were performed by flow cytometry, alkaline phosphatase and mineralization stains, RT-PCR, immunohistochemistry, scanning electron microscopy, Western blot and enzymatic activity. Moreover, the genetic stability was evaluated and compared before and after differentiation by short-comparative genomic hybridization (sCGH). RESULTS: DPPSC showed excellent differentiation into osteogenic lineages expressing bone-related markers similar to SAOS-2. When cells were cultured on biomaterials, DPPSC showed higher initial adhesion levels. Nevertheless, their osteogenic differentiation showed similar trend among both cell types. Interestingly, only DPPSC maintained a normal chromosomal dosage before and after differentiation on 2D monolayer and on biomaterials. CONCLUSIONS: Taken together, these results promote the use of DPPSC as a new pluripotent-like cell model to evaluate the biocompatibility and the differentiation capacity of biomaterials used in bone regeneration.

Does smoking affect the Ki67 and p53 expressions in asymptomatic fully impacted lower third molar follicles?

PURPOSE: Ki67 and p53 protein expressions are the most widely used markers to show the pathologic proliferation and early-stage tumoral alterations in vital tissues. The aim of this study was to compare Ki67 and p53 protein expressions in smokers' and nonsmokers' pericoronal follicles of asymptomatic impacted lower third molars (ILTMs). MATERIALS AND METHODS: A cross-sectional study was planned. The study sample was derived from a population of patients who presented for evaluation and operative treatment of asymptomatic ILTMs. The predictor variable was smoking status, defined as smoker or nonsmoker. Outcome variables were Ki67 and p53 protein expressions in ILTM follicles. Other study variables were age, gender, tooth position, cigarette pack-year, epithelial layer staining, and inflammation. Independent-samples t test analyses were conducted with SPSS 10.0 (SPSS, Inc, Chicago, IL), with statistical significance set at a P value equal to .05. RESULTS: The study sample was composed of 70 patients (35 in the smoker group) who contributed 60 follicles. There were statistical differences between the 2 groups for variables Ki67 and p53. Mean expression levels of Ki67 were 3.93 ± 2.17 and 2.48 ± 2.09, respectively, for smokers and nonsmokers (P = .011). Mean expression levels of p53 were 5.32 ± 1.98 and 3.06 ± 2.34, respectively, for smokers and nonsmokers (P = .000). CONCLUSION: The present study showed that dental follicles of smokers have higher Ki67 and p53 protein expressions than nonsmokers' follicles.

Dental pulp of the third molar: a new source of pluripotent-like stem cells.

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However, no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work, we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF, EGF and PDGF. These cells are SSEA4(+), OCT3/4(+), NANOG(+), SOX2(+), LIN28(+), CD13(+), CD105(+), CD34(-), CD45(-), CD90(+), CD29(+), CD73(+), STRO1(+) and CD146(-), and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly, DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm, endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4, GATA6, MIXL1, NANOG, OCT3/4, SOX1 and SOX2 to determine the degree of similarity between DPPSCs, EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs, hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages, they represent an easily accessible source of stem cells, which opens a range of new possibilities for regenerative medicine.

Expression of the stem cell marker, SOX2, in ameloblastoma and dental epithelium.

Ameloblastomas are locally invasive odontogenic tumors that exhibit a high rate of recurrence and often associate with the third molars. They are suggested to originate from dental epithelium because the tumor cells resemble epithelial cells of developing teeth. Expression of the transcription factor SOX2 has been previously localized in epithelial stem and progenitor cells in developing teeth as well as in various tumors. Here, we show that SOX2 is expressed in the epithelial cells of follicular and plexiform ameloblastomas. SOX2 was localized in the dental lamina of developing human primary molars. It was also expressed in the fragmented dental lamina associated with the third molars and in the epithelium budding from its posterior aspect in mice. However, no SOX2 expression was detected in either Hertwig's epithelial root sheath directing the formation of roots or in the epithelial cell rests of Malassez covering the completed roots. SOX2 was associated with supernumerary tooth formation in odontoma-like tumors induced by Wnt signal activation in mice. We propose that SOX2 functions in maintaining the progenitor state of epithelium in ameloblastomas and that ameloblastomas may originate from SOX2-expressing dental lamina epithelium.

Leptin expression in healthy and inflamed human dental pulp.

AIM: To investigate the expression of leptin in healthy and inflamed human dental pulp. METHODOLOGY: Twenty-one pulp samples were obtained from freshly caries- and restoration-free extracted human third molars. In seven-third molars (inflamed pulp group), inflammation was induced prior to extraction. Pulp samples were processed, and leptin expression was determined by quantitative real-time PCR (qRT-PCR) and the amount of leptin by immunoblot. RESULTS: All healthy and inflamed dental pulp samples expressed leptin. Western blot analysis revealed the presence of a protein with an apparent molecular weight of ~16 kDa in human dental pulp, which corresponds to the estimated molecular weight of leptin. The expression of leptin mRNA in dental pulp was confirmed by qRT-PCR analysis, and the size of the amplified fragments (296 bp for leptin and 194 bp for cyclophilin) was confirmed by agarose gel electrophoresis. The expression of leptin in the inflamed pulp group was significantly greater (P < 0.05) than in healthy teeth. The relative amount of leptin in inflamed pulps was almost twice than in healthy pulps. CONCLUSIONS: For the first time, the presence of leptin in human dental pulp tissues has been demonstrated. The upregulation of leptin expression in inflamed pulp samples suggests that leptin can play a role in pulpal inflammatory and immune responses.

Direct measurement of stain retention in third molars.

AIM: To directly determine the mass of dye retained in teeth following exposure to aqueous solutions of Rhodamine B and to correlate tooth color modifications. MATERIALS AND METHODS: Extracted third molars (25) were selected and sectioned at the cementoenamel junction for coronal staining. Pulp tissue was removed and teeth sonicated to remove debris. Teeth were kept in deionized water for 12 hours and subsequently weighed. They were then stained for 4 hours in 5 ml of Rhodamine B dye at two different concentrations. The samples were then subjected to two 8 hours rinses in deionized water. The tooth shade was recorded with a commercially available intraoral spectrophotometer (Vita Easyshade Compact, Vita Zahnfabrik, Bad Säckingen, Germany) at baseline (T1), after dye immersion (T2), and after water rinsing (T3). A standard absorption curve was then used to calculate the dye mass in the rinse solutions as well as the post- treatment stain solutions. All solution optical absorption curves were recorded using a laboratory research spectrophotometer (Cary 300, Agilent, USA). The mass of dye in each solution was then calculated from the standard curve relating optical absorption to aqueous dye concentration. RESULTS: An average change in the CIE (a) values of 8.0 ± 0.3 were observed for concentrations of Rhodamine B similar to the optical appearance of wine or other darkly colored juices while an increase of 10× in concentration gave values too high to measure using a standard intraoral spectrophotometer. By measuring the optical absorbance of the staining solutions before and after the staining process, we were able to measure dye retention of 54 ± 26 micrograms per gram of tooth. CONCLUSION: While no significant correlation could be found between the amount of stain retention in the dentition and the tooth shade due to the high uncertainties in the spectroscopic measurements, we were able to show that this method should admit such comparisons for future research. CLINICAL SIGNIFICANCE: The development of a reliable chromophore infiltration model may provide standardized and reproducible results in evaluating tooth whitening efficacy.

Combination of plant phenolics and isoquinolinium alkaloids protects gingival fibroblast and improves post-extraction healing after lower third molar extraction.

AIMS: The effect of polyphenolic fraction of Lonicera caerulea (PFLC) and alkaloid fraction of Macleaya cordata (AFMC) mix on the production of inflammatory mediators in human gingival fibroblasts pretreated with lipopolysaccharide (LPS) was investigated. In addition, protective effects of mucoadhesive paste containing combination of PFLC and AFMC (0.05% and 0.01%, respectively; n=15, Group A) and placebo (n=15, Group B) were evaluated in patients after surgical extraction of lower third molars. METHODS: Gingival fibroblasts were pre-treated with LPS (10 µg/mL; 24 h) and PFLC/AFMC (25/0.25; 50/0.25; 100/0.25; 25/0.5; 50/0.5; 100/0.5 µg/mL) in serum-free medium was applied for 4 h. Then the interleukin-6 (IL-6), reactive oxygen species (ROS) generation, level of intracellular glutathione (GSH) and expression of cyclooxygenase-2 (COX-2) were evaluated. The study was a 6-day, single-center, randomized, double-blind and placebo-controlled trial consisting of two parallel treatment arms. A modified Oral health impact profile questionnaire including both general oral condition and extraction related questions, was used to evaluate the oral condition and other changes before (day 0) and on the days 1, 3 and 6 after surgical extraction. RESULTS AND CONCLUSION: The combination of PFLC with AFMC caused a reduction of ROS generation, reduced IL-6 production and suppressed the expression of COX-2. In group A the paste treatment contributed to improvement of oral health-related quality of life. Topical application of PFLC and AFMC into the extraction wound improved post-extraction site wound healing probably by antioxidant and anti-inflammatory mechanisms.

Malondialdehyde levels in dental follicles of asymptomatic impacted third molars.

PURPOSE: Increased levels of reactive oxygen species lead to oxidative stress and tissue damage. Malondialdehyde (MDA) is one of many low-molecular-weight endproducts of lipid peroxidation that increases with oxidative stress. The aim of this study was to determine oxidative stress in dental follicles (DFs) of radiologically asymptomatic impacted third molars (ITMs) using MDA. MATERIALS AND METHODS: This study involved 40 DFs of 40 patients referred for clinically and radiographically asymptomatic ITMs. Forty healthy gingival tissues in the same patients were obtained during surgical removal of teeth as a control group. DF widths on periapical radiographs narrower than 2.5 mm were included in the study. All tissues samples were analyzed for MDA as an indicator of oxidative stress. RESULTS: Levels of MDA were significantly higher in DFs from ITMs than those from healthy gingival tissues of the same patients (P < .01). CONCLUSION: The results suggest that significant oxidative stress may occur in DFs of asymptomatic ITMs. The findings suggest that increased MDA may play an important role in oxidative stress in DFs. In light of these preliminary findings of the present study, further investigations and comprehensive studies are required to determine the role of antioxidants that scavenge free radicals in DFs.

Gene Expression of Vascular Endothelial Growth Factor A and its Receptors in Dental Pulp of Immature and Mature Teeth.

OBJECTIVE: Vascular endothelial growth factor A (VEGFA) and its receptors are essential proteins for the angiogenic activity of dental pulp. Angiogenesis fundamentally provides oxygen and nutrients to cells for root formation and defence mechanisms. The angiogenic potential of dental pulp should be understood and considered for the conservative and regenerative endodontics. The purpose of this research was to measure the VEGFA expression and its receptors such as vascular endothelial growth factor receptors 1, -2 (VEGFR1, VEGFR2) and Neuropilin 1 (NRP1) in human dental pulp from molars with immature and mature apexes. METHODS: VEGFA system mRNAs expressions were assessed in dental pulp obtained from freshly extracted human third molars divided into immature (n=8) and mature (n=8) apexes. RNAs were extracted from the samples. Each sample's cDNA was synthetized and the target genes VEGFA, VEGFR1, VEGFR2, NRP1 expression profiles obtained by RT2-PCR. Analysis was based on the Student's t-test comparing the replicate 2-ΔCt values for each gene. P values of <0.05 were considered significant. RESULTS: In teeth with mature apexes, VEGFA (P=0.0002), NRP1 (P=0.0001), VEGFR1 (P=0.0057) and VEGFR2 (P=0.018259) significantly increased statistically with respect to the immature apexes group. CONCLUSION: Within the limitation of the present investigation, it can be concluded that the angiogenic process seems to be a physiological process in the dental pulp due to the studied angiogenic growth factor are expressed in both immature and mature dental pulps. VEGFA and its receptors are expressed significantly higher in mature apex teeth than immature apex teeth.

Pathological changes and immunoexpression of p63 gene in dental follicles of asymptomatic impacted lower third molars: an immunohistochemical study.

OBJECTIVE: The aim of this study was to examine the pathologic changes and immunoexpressivity of p63 gene in dental follicles (DFs) of asymptomatic partially and completely impacted lower third molars. STUDY DESIGN: Clinical and radiologic examinations included 50 DFs with no signs of abnormal radiolucency (follicular space <2.5 mm), taken from 50 patients. RESULTS: Histopathologic examinations of the specimens revealed 47 normal dental follicular tissues, 1 ameloblastoma, and 2 dentigerous cysts. p63 Immunoexpressivity was stronger in the DFs of the group with completely impacted teeth (64%),than it was in the case of DFs of the group with partially impacted teeth (40%). CONCLUSIONS: Stronger p63 gene immunoexpression in the group with completely impacted teeth might be a consequence of bigger number of stem cells than it is in the case of the group with partially impacted teeth. This study also supports prophylactic removal of impacted teeth because of the development of pathologies associated with them.

Molecular characterization of human impacted third molars: diversification of compartments.

To gain more insight into the development of human teeth, we characterized different compartments of impacted third molars at two developmental stages by assessing expression levels of a set of genes. We considered genes known to be essential for the development of teeth and ectomesenchyme as well as genes covering characteristic features of stemness. Molars were divided into the operculum, periodontal ligament, developing pulp and, using a new approach, the pad-like tissue beneath the developing pulp. Markers for ectomesenchyme and tooth development known from rodents were assayed by semiquantitative PCR and every compartment was assigned its own signature of gene expression. The expression of markers characteristic of stem cells pointed to multipotent features. The expression patterns found shift in the course of development underscoring the relevance of these genes involved in human tooth development. The results suggest an inherent asymmetry between the developing pulp and pad-like tissue established early in tooth development. A microarray analysis of cells derived from pad-like tissue and pulp proper was performed to obtain cues regarding the consequences of tissue diversification. Both sets of data support the validity of our new approach to the subdivision of the developing tooth, by indicating a compartment-dependent commitment of isolated cells probably due to the postulated asymmetry within the developing tooth germ.

The hidden structure of human enamel.

Enamel is the hardest and most resilient tissue in the human body. Enamel includes morphologically aligned, parallel, ∼50 nm wide, microns-long nanocrystals, bundled either into 5-µm-wide rods or their space-filling interrod. The orientation of enamel crystals, however, is poorly understood. Here we show that the crystalline c-axes are homogenously oriented in interrod crystals across most of the enamel layer thickness. Within each rod crystals are not co-oriented with one another or with the long axis of the rod, as previously assumed: the c-axes of adjacent nanocrystals are most frequently mis-oriented by 1°-30°, and this orientation within each rod gradually changes, with an overall angle spread that is never zero, but varies between 30°-90° within one rod. Molecular dynamics simulations demonstrate that the observed mis-orientations of adjacent crystals induce crack deflection. This toughening mechanism contributes to the unique resilience of enamel, which lasts a lifetime under extreme physical and chemical challenges.

In vitro remineralization of artificial enamel caries with resin composites containing calcium phosphate particles.

The aim of the study was to evaluate the effect of experimental composites containing dicalcium phosphate dihydrate (DCPD) on remineralization of enamel lesions. Five resin-based composites containing equal parts (in mols) of bisphenol-A glycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA), and 60 vol % of fillers were manipulated. Filler phase was constituted by silanized barium glass and 0, 10, or 20 vol % of DPCD particles, either functionalized (F) or nonfunctionalized (NF) with TEGDMA. Artificial subsurface lesions were produced in human enamel fragments and divided according to the resin composite applied on the lesion (no DCPD, 20% NF, 20% F, 10% NF, 10% F) plus a group without composite build-up (nontreated, NT). Fragments were exposed to 16 days of pH cycling. Specimens were evaluated using transverse microradiography (TMR). Calcium and phosphate concentrations in pH-cycling solutions were determined by spectrophotometry. TMR and ionic concentrations were analyzed using one-way ANOVA/Tukey and Kruskal-Wallis/Dunn test, respectively (alpha: 0.05). All composite groups showed enamel remineralization (3%-23%). Higher mineral recovery in the middle (7%-11%) and bottom (2%-7%) thirds of the lesion was observed in groups with DCPD-containing composites compared to the "no DCPD" group (middle: 1%, bottom: -3%). Lesion depth was significantly reduced in groups using DCPD-containing composites compared to NT group. No noticeable increase in calcium and phosphate ions was observed in the pH-cycling solutions due to the presence of DCPD in the composites. In conclusion, composites with DCPD fractions as low as 10%, regardless of functionalization, were able to promote mineral recovery and reduce lesion depth of enamel lesions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1542-1550, 2019.

Mitochondrial genome mutations in mesenchymal stem cells derived from human dental induced pluripotent stem cells.

Ethical and safety issues have rendered mesenchymal stem cells (MSCs) popular candidates in regenerative medicine, but their therapeutic capacity is lower than that of induced pluripotent stem cells (iPSCs). This study compared original, dental tissue-derived MSCs with re-differentiated MSCs from iPSCs (iPS-MSCs). CD marker expression in iPS-MSCs was similar to original MSCs. iPS-MSCs expressed higher in pluripotent genes, but lower levels in mesodermal genes than MSCs. In addition, iPS-MSCs did not form teratomas. All iPSCs carried mtDNA mutations; some shared with original MSCs and others not previously detected therein. Shared mutations were synonymous, while novel mutations were non-synonymous or located on RNA-encoding genes. iPS-MSCs also harbored mtDNA mutations transmitted from iPSCs. Selected iPS-MSCs displayed lower mitochondrial respiration than original MSCs. In conclusion, screening for mtDNA mutations in iPSC lines for iPS-MSCs can identify mutation-free cell lines for therapeutic applications. [BMB Reports 2019; 52(12): 689-694].

Characterization of dental pulp stem cells of human tooth germs.

In previous studies, human dental pulp stem cells (hDPSCs) were mainly isolated from adults. In this present study, we characterized hDPSCs isolated from an earlier developmental stage to evaluate the potential usage of these cells for tissue-regenerative therapy. hDPSCs isolated at the crown-completed stage showed a higher proliferation rate than those isolated at a later stage. When the cells from either group were cultured in medium promoting differentiation toward cells of the osteo/odontoblastic lineage, both became alkaline-phosphatase-positive, produced calcified matrix, and were also capable of forming dentin-like matrix on scaffolds in vivo. However, during long-term passage, these cells underwent a change in morphology and lost their differentiation ability. The results of a DNA array experiment showed that the expression of several genes, such as WNT16, was markedly changed with an increasing number of passages, which might have caused the loss of their characteristics as hDPSCs.

Population pharmacokinetics of articaine with 1:200,000 epinephrine during third molar surgery and simulation of high-dose regimens.

BACKGROUND AND OBJECTIVES: Articaine is more and more used in third molar surgery under local anesthesia (LA). The objectives of this analysis were to characterize the pharmacokinetics of articaine for this type of surgery and to simulate dosing regimens. METHODS: Non-linear mixed-effects modeling conducted in Monolix 4.4.0 was used to describe articaine plasma concentration-time data from 20 patients. Monte Carlo simulations were then performed to evaluate the probability of cardiotoxic target attainment (PCTA) of various dosage regimens. RESULTS: Articaine concentration data were best described by a linear one-compartment model, with an additional depot compartment for submucosal route with a zero-order transfer to central compartment. Age and gender were found to influence duration transfer (Tk0) and elimination rate constant (Ke), respectively. Simulated maximum recommended dose regimen (7mg/kg) had a PCTA of 0%. Simulated higher doses of 10mg/kg and 15mg/kg had a PCTA of 0% and about 1-4%, respectively. CONCLUSIONS: The model adequately described the articaine pharmacokinetics. This is the first PK model qualified for articaine administered by submucosal route. The simulations suggest that maximum recommended dose regimen is safe concerning the cardiotoxicity in healthy patients.

Effects of TGF-beta 1 on interleukin profile of human dental pulp and odontoblasts.

Transforming growth factor-beta 1 (TGF-beta1) is the most extensively studied growth factor in dentin-pulp complex, with pleiotropic effects on pulp response and healing. Our main objective was to analyze the expression profile of pulp tissue and odontoblasts, and the effects of TGF-beta1 on these profiles in cultured human pulp and odontoblasts with a specific interest in the anti- and pro-inflammatory cytokines. For that purpose, pulps and odontoblasts were cultured for different time periods, and microarray was performed to both cultured and native samples. Of cytokines, various interleukins (IL) were confirmed by RT-PCR, and in +/- TGF-beta1 treated pulps also by antibody array. Pro-inflammatory IL-7, -12alpha and -16 mRNAs were detected in native pulp. The expression levels of pro-inflammatory IL-1alpha, -1beta, -6 and -8 were clearly induced after TGF-beta1 treatment, while no anti-inflammatory cytokines were induced. Of all pulpal interleukins analyzed IL-6 and -8 were present at the highest levels in conditioned pulp tissue media. In native odontoblasts pro-inflammatory IL-6 and -7 mRNAs were detected, and in cultured odontoblasts pro-inflammatory IL-8 mRNA showed over 20-fold transient induction after TGF-beta1 treatment. Our results demonstrate that TGF-beta1 is a potent regulator of pro-inflammatory responses and defensive reactions in dentin-pulp complex.

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.