Anibal Diogenes

Evaluation of survival, proliferation and neurodifferentiation of dental stem cells from the apical papilla (SCAP) in fibrin hydrogels. We hypothesized that fibrin composition will influence cell behavior. Modulus, pore and fiber size were measured. SCAP in vitro viability, proliferation and neural differentiation, as well as in vivo proliferation and angiogenesis were studied. Hydrogel moduli were influenced by fibrin formulation but not hydrogel morphology, SCAP in vitro viability and proliferation. In total 60% of SCAP expressed PanNeurofilament in vitro without induction in Fibrinogen50-Thrombin10. SCAP proliferated when implanted in vivo and stimulated host endothelial cell infiltration. Fibrinogen30-Thrombin10 or Thrombin50 would be more favorable to in vitro SCAP viability and in vivo proliferation, while Fibrinogen 50-Thrombin50 would be more adapted to neurodifferentiation.

ABSTRACT Objective: To compare the phenotype, the growth curve and the osteo- and chondrogenic differentiation potential of mixed vs purified stem cells populations from the apical papilla (SCAPs); gingival fibroblasts (GFs) were used as control. Method: Four cell populations were considered: 1)mixed SCAPs (M-SCAPs); 2&3)SCAPs purified by immunomagnetic separation based either on Stro-1 (P-SCAPs1), or on a combination of CD73, CD90 and CD105 (P-SCAPs2); 4)gingival fibroblasts (GFs). Each population was subcultured, and their growth curve determined. Their phenotype was characterized by flow cytometry using antibodies against CD29, CD31, CD34, CD90, CD105 and Stro-1. The cells were then cultured in osteo- and chondrogenic media during 25 days. Osteogenic and chondrogenic differentiation were evaluated respectively by alizarin red S and alcian blue staining. Result: All four cell populations had similar growth curves, were homogeneously positive to CD29, CD90 and CD105 (percentage of positive cells >97% in all cases but P-SCAPs1 for CD90 – 91.5%), and negative to CD31, CD34 and Stro-1 (<3% in all cases but GFs for CD34 – 7.5%). All cell populations were positive for alizarin red S, and all but GFs were positive for alcian blue. Conclusion: The present results question the specificity of certain markers considered to identify dental mesenchymal stem cells. The expression of Stro-1 was notably lost in P-SCAPs1, which however conserved their multi-differentiation potential. The rationale of sorting cells based on these markers is therefore called into question vs using a mixed population. Finally, the results suggest that GFs either contain a certain proportion of osteogenic progenitor cells, or that they intrinsically possess an osteogenic differentiation potential.

Recent studies have demonstrated that the lipopolysaccharide (LPS) receptor (TLR4) is expressed in TRPV1 containing trigeminal sensory neurons. In this study, we evaluated whether LPS activates trigeminal neurons, and sensitizes TRPV1 responses via TLR4. To test this novel hypothesis, we first demonstrated that LPS binds to receptors in trigeminal neurons using competitive binding. Second, we demonstrated that LPS evoked a concentration-dependent increase in intracellular calcium accumulation (Ca)i and inward currents. Third, LPS significantly sensitized TRPV1 to capsaicin measured by (Ca)i, release of calcitonin gene-related peptide, and inward currents. Importantly, a selective TLR4 antagonist blocked these effects. Analysis of these data, collectively, demonstrates that LPS is capable of directly activating trigeminal neurons, and sensitizing TRPV1 via a TLR4-mediated mechanism. These findings are consistent with the hypothesis that trigeminal neurons are capable of detecting pat...

Regenerative endodontic procedures (REPs) have emerged as a treatment option for immature necrotic teeth to allow the reestablishment of a newly formed vital tissue and enable continued root development. The apical papilla stem cells (SCAPs) play an important role in physiologic root development and may also contribute to further root development during REPs. The goal of these case reports is to show evidence of the apical papilla survival and development, in human teeth with apical periodontitis, after REPs, with 5-year clinical and radiographic follow-up. In the first case, an 11-year-old girl with acute apical abscess of tooth 15 was referred for a REP. Treatment was performed with an intracanal medication followed by induction of a blood clot and a Mineral Trioxide Aggregate (MTA) cervical barrier. The 5-year follow-up showed an appreciable increase in root length as well as root canal thickness. In case 2, a 16-year-old girl was referred for endodontic treatment of tooth 21. Th...

Cone-beam computed tomography (CBCT) has promoted changes in approaches in Endodontics, and enhanced decision-making in complex clinical cases. Despite the technological advancements in CBCT hardware, the interpretation of the acquired images is still compromised by viewing software packages that often have limited navigational tools and lack adequate filters to overcome some challenges of the CBCT technology such as artefacts. This study reviews the current limitations of CBCT and the potential of a new CBCT software package (e-Vol DX, CDT- Brazil) to overcome these aspects and support diagnosing, planning and managing of endodontic cases. This imaging method provide high resolution images due to submillimeter voxel sizes, dynamic multi-plane imaging navigation and ability to change the volume parameters such as slice thickness and slice intervals and data correction applying imaging filters and manipulating brightness and contrast. The main differences between e-Vol DX and other s...

The purpose of this study was to determine the position of the apical foramen (AF) in relation to root surfaces of human permanent teeth using cone-beam computed tomographic (CBCT) imaging and novel advanced imaging analysis software (e-Vol DX; CDT Software, Bauru, SP, Brazil). The AF position was determined on CBCT scans viewed and analyzed using e-Vol DX of 1400 teeth (422 patients) according to the root surface as follows: buccal, mesiobuccal, mesial, mesiolingual/palatal, lingual/palatal, distolingual/palatal, distal, distobuccal, and central. Categoric variables were described as frequencies and percentages. Frequencies were reported with their confidence intervals (95%). Categoric variables were analyzed using the chi-square test with Yates correction. The level of significance was set at α = 0.05. The most frequent AF position in maxillary anterior teeth was central (46%-60%). The AF in mandibular central incisors was buccal in 44% of the cases. In maxillary first and second ...

This randomized clinical study compared the antibacterial effectiveness of treatment protocols using either a triple antibiotic solution (1 mg/mL) or calcium hydroxide/chlorhexidine paste as interappointment medication in infected canals of teeth with primary apical periodontitis. The root canals of single-rooted teeth with apical periodontitis were prepared by using a reciprocating single-instrument technique with 2.5% sodium hypochlorite irrigation and then medicated for 1 week with either a triple antibiotic solution (minocycline, metronidazole, and ciprofloxacin) at 1 mg/mL (n = 24) or a calcium hydroxide paste in 2% chlorhexidine gluconate (n = 23). Samples were taken from the canal at the baseline (S1), after chemomechanical preparation (S2), and after intracanal medication (S3). DNA extracts from clinical samples were evaluated for total bacterial reduction using a 16S ribosomal RNA gene-based quantitative polymerase chain reaction assay. All S1 samples were positive for the ...

Treatment of immature permanent teeth with necrotic pulp and apical pathosis constitutes a challenge for endodontists. The present study was done to evaluate the effect of age and apical diameter on the regenerative potential of young permanent immature teeth with necrotic pulps. Immature necrotic permanent maxillary incisors (n = 40) of patients 9-18 years old were divided into 2 groups according to the treatment protocol: group Y (younger age group), 9-13 years and group O (older age group), 14-18 years. Each group was further subdivided into 2 subgroups according to apical diameter, subgroup (n) (narrower diameter) between 0.5 and 1 mm and subgroup (w) (wider diameter) equal to or greater than 1 mm. Revascularization procedures were performed for all patients. Follow-up was done for up to 12 months. Standardized radiographs were digitally evaluated for increase in root length and thickness and decrease in apical diameter. After the follow-up period, most of the cases demonstrated radiographic evidence of periapical healing. Group Y showed significant progressive increase in root length and width and decrease in apical diameter. Subgroup (w) representing wider apical diameter showed significant progress as well. It was found that revascularization procedures can be implemented in any age ranging from 9 to 18 years; however, younger age groups were better candidates for revascularization procedure than older ones. Regarding the apical diameter, regeneration procedures were successful with apical diameters as small as 0.5 mm. However, teeth with preoperative wider diameters (≥1 mm) demonstrated greater increase in root thickness, length, and apical narrowing.

Contemporary endodontics has seen an unprecedented advance in technology and materials. This article aimed to review some of the challenges and advances in the following sections: (1) endodontic imaging, (2) root canal preparation, (3) root canal disinfection, (4) root canal filling, and (4) regenerative endodontic procedures (REPs). Jointly, these advances are aimed at improving the state of the art and science of root canal treatment.

The development of regenerative endodontic therapies offers exciting opportunities for future improvements in treatment outcomes. Advances in our understanding of regenerative events at the molecular and cellular levels are helping to underpin development of these therapies, although the various strategies differ in the translational challenges they pose. The identification of a variety of bioactive molecules, including growth factors, cytokines, chemokines, and matrix molecules, sequestered within dentin and dental pulp provides the opportunity to present key signaling molecules promoting reparative and regenerative events after injury. The protection of the biological activity of these molecules by mineral in dentin before their release allows a continuing supply of these molecules, while avoiding the short half-life and the non-human origin of exogenous molecules. The ready release of these bioactive molecules by the various tissue preparation agents, medicaments, and materials c...

The spatial and temporal control of stem cell differentiation into odontoblast-like cells remains one of the major challenges in regenerative endodontic procedures. The current study aims to synthesize and compare the effect of dexamethasone (Dex) release from 2 variants of Dex-loaded chitosan nanoparticles (CSnp) on the odontogenic differentiation of stem cells from apical papilla (SCAP). Two variants of Dex-loaded CSnp were synthesized by encapsulation (Dex-CSnpI) and adsorption (Dex-CSnpII) methods. The physicochemical characterization of Dex-CSnpI and Dex-CSnpII was assessed by transmission electron microscopy, Zetasizer, and Fourier transform infrared spectroscopy, whereas the Dex release kinetics was assessed by spectrophotometry. A previously characterized SCAP cell line was cultured onto CSnp, Dex-CSnpI, or Dex-CSnpII. The biomineralization potential was determined by alizarin red staining. Alkaline phosphatase, dentin sialophosphoprotein, and dentin matrix protein-1 gene expressions were analyzed by real-time reverse-transcription polymerase chain reaction. Dex-CSnpI resulted in slower release of Dex compared with Dex-CSnpII, but both demonstrated sustained release of Dex for 4 weeks. Biomineralization of SCAP was significantly higher (P < .05) in presence of Dex-CSnpII compared with that in Dex-CSnpI at 3 weeks. Alkaline phosphatase gene expression was significantly higher in the presence of Dex-CSnpII compared with Dex-CSnpI, with peak expression seen at 2 weeks (P < .05). The expression of odontogenic specific marker dentin matrix protein-1 was significantly higher in presence of Dex-CSnpII compared with Dex-CSnpI at 3 weeks (P < .05). Collectively, these data suggest that sustained release of Dex results in enhanced odontogenic differentiation of SCAP. These findings highlight the potential of temporal-controlled delivery of bioactive molecules to direct the spatial- and temporal-controlled odontogenic differentiation of dental stem cells.

Stem cells from the apical papilla (SCAP) are a population of mesenchymal stem cells likely involved in regenerative endodontic procedures and have potential use as therapeutic agents in other tissues. In these situations, SCAP are exposed to hypoxic conditions either within a root canal devoid of an adequate blood supply or in a scaffold material immediately after implantation. However, the effect of hypoxia on SCAP proliferation and differentiation is largely unknown. Therefore, the objective of this study was to evaluate the effect of hypoxia on the fate of SCAP. SCAP were cultured under normoxia (21% O2) or hypoxia (1% O2) in basal or differentiation media. Cellular proliferation, gene expression, differentiation, and protein secretion were analyzed by live imaging, quantitative reverse-transcriptase polymerase chain reaction, cellular staining, and enzyme-linked immunosorbent assay, respectively. Hypoxia had no effect on SCAP proliferation, but it evoked the up-regulation of genes specific for osteogenic differentiation (runt-related transcription factor 2, alkaline phosphatase, and transforming growth factor-β1), neuronal differentiation ( 2'-3'-cyclic nucleotide 3' phosphodiesterase, SNAIL, neuronspecific enolase, glial cell-derived neurotrophic factor and neurotrophin 3), and angiogenesis (vascular endothelial growth factor A and B). Hypoxia also increased the sustained production of VEGFa by SCAP. Moreover, hypoxia augmented the neuronal differentiation of SCAP in the presence of differentiation exogenous factors as detected by the up-regulation of NSE, VEGFB, and GDNF and the expression of neuronal markers (PanF and NeuN). This study shows that hypoxia induces spontaneous differentiation of SCAP into osteogenic and neurogenic lineages while maintaining the release of the proangiogenic factor VEGFa. This highlights the potential of SCAP to promote pulp-dentin regeneration. Moreover, SCAP may represent potential therapeutic agents for neurodegenerative conditions because of their robust differentiation potential.

Regenerative endodontic procedures are an alternative treatment for immature teeth with necrotic pulps. Typically, intracanal medicaments such as triple antibiotic paste (TAP) or double antibiotic paste (DAP) and calcium hydroxide (Ca[OH](2)) are used for disinfection. However, their effect on human stem cells of the apical papilla (SCAPs) is unknown. We hypothesized that intracanal medicaments at high concentrations are toxic to SCAPs. To test this hypothesis, a cell culture assay was used. Briefly, SCAPs were cultured and subjected to either no drug treatment or various concentrations including TAP, DAP, modified TAP (ciprofloxacin, metronidazole and cefaclor), Augmentin (Champs Pharmacy, San Antonio, TX), or Ca(OH)(2). Viable stem cells counts were obtained using an automated method of detecting trypan blue dye at 3 days after treatment. All 4 antibiotics significantly reduced SCAP survival in a concentration-dependent fashion. Interestingly, Ca(OH)(2) was conducive with SCAP survival at all concentrations. Collectively, our data show that high concentrations of antibiotics have a detrimental effect on SCAP survival, whereas lower concentrations as well as Ca(OH)(2) at all tested concentrations are conducive with SCAP survival and proliferation. These studies highlight the clinically important point that intracanal medicaments must be used at concentrations that are bactericidal while having minimal effects on stem cell viability.

Recent studies have demonstrated that the lipopolysaccharide (LPS) receptor (TLR4) is expressed in TRPV1 containing trigeminal sensory neurons. In this study, we evaluated whether LPS activates trigeminal neurons, and sensitizes TRPV1 responses via TLR4. To test this novel hypothesis, we first demonstrated that LPS binds to receptors in trigeminal neurons using competitive binding. Second, we demonstrated that LPS evoked aconcentration-dependent increase in intracellular calcium accumulation (Ca2+)i and inward currents. Third, LPS significantly sensitized TRPV1 to capsaicin measured by (Ca2+)i, release of calcitonin gene-related peptide, and inward currents. Importantly, a selective TLR4 antagonist blocked these effects. Analysis of these data, collectively, demonstrates that LPS is capable of directly activating trigeminal neurons, and sensitizing TRPV1 via a TLR4-mediated mechanism. These findings are consistent with the hypothesis that trigeminal neurons are capable of detecting pathogenic bacterial components leading to sensitization of TRPV1, possibly contributing to the inflammatory pain often observed in bacterial infections.

The role of protease activated receptor-2 (PAR-2) activation in trigeminal nociception and in induction of functional competence in the delta opioid receptor (DOR) is not known. In this study, we evaluated whether agonists of PAR-2 activate the capsaicin-sensitive subclass of trigeminal nociceptors in a PLC-PKC-dependent manner and induce functional competence in the DOR. Adult male rat trigeminal ganglion (TG) cultured neurons were treated with the PAR-2 agonist (SL-NH2) or an enzyme activator of PAR (trypsin) and the activation of TG nociceptors was assessed using three independent methods: neuropeptide release, calcium influx, and whole cell patch-clamp. The specificity of SL-NH2 and trypsin responses was evaluated using TG cultures transfected with siRNA against PAR-2. The in vivo role of PAR-2 activation was determined measuring SL-NH2 and trypsin-evoked nocifensive behavior and increase in blood flow. Trigeminal neurons were treated with SL-NH2/vehicle and then the DOR agonist to determine DOR inhibition of evoked neuropeptide release and cAMP accumulation. The results showed that SL-NH2 (100 microM) and trypsin (1-600 nM) activate TG nociceptors, which is partly reversible by the PKC inhibitor bisindolylmaleimide (500 nM) and by ruthenium red (10 microM). In cultures treated with siRNA against PAR-2, both SL-NH2 and trypsin responses were significantly diminished. Both SL-NH2 and trypsin evoke nocifensive behavior and increases in blood flow in an orofacial pain model. Application of SL-NH2 rapidly produced functional competence of DOR for inhibiting nociceptor function. In inflamed tissue, endogenous proteases may activate TG nociceptors and generate pain. Moreover, activation of PAR-2 can also induce functional competence in DOR.

Galanin (GAL) has been implicated in modulating anxiety, although a precise role remains unclear. Previous studies revealed anxiolytic effects, anxiogenic effects, or no effect, depending on the test, brain region, route of drug administration and context. We have shown ...

Stem cells of the apical papilla (SCAP) have been identified as an important population of mesenchymal stem cells (MSCs) in regenerative endodontics. Preclinical studies that evaluate the various aspects of the regenerative process must use fully characterized MSCs. The phenotype of these cells when maintained in culture is crucial for the translational applicability of these studies. Thus, in this study, we aimed to characterize a SCAP cell line that preferentially expressed and maintained the required MSCs markers in culture, namely the RP-89 cell line. Apical papillae from extracted mandibular third molars from a single donor were processed for immunohistochemistry, cell culture, and RT-PCR. SCAP were successfully cultured and maintained in culture for up to 20 passages. The expression of MSC-related molecular markers was analyzed by laser confocal microscopy, flow cytometry, and real-time RT-PCR arrays. Cells within the apical papillae tissue had a widespread expression of CD90, whereas the expression of CD105 and CD73 was compartmentalized to the blood vessels and periphery, respectively. The RP-89 cell line population coexpressed CD73, CD90, and CD105 in all passages evaluated. There was a dramatic change in gene expression when cells were cultured and maintained in culture with the up-regulation of MSCs markers, inhibitors of differentiation, and stemness markers. Conversely, genes involved in the differentiation of MSCs were suppressed. Collectively, these results highlight the need to use fully characterized cell lines in regenerative studies and provide the foundational knowledge of gene expression modulation that occurs in cultured SCAP.