Roots Of Human Teeth Research Articles

Geomorphologic study of human tooth root surfaces following simulated avulsion by scanning electron microscopy.

Tooth avulsion is the most severe traumatic tooth injury. Immediate replantation after avulsion provides the highest success rate, whereas delayed replantation with dehydration for >1h always results in ankylosis, replacement resorption, and eventual tooth loss. The aim of this study was to investigate the geomorphologic change of ultrastructure on tooth root surfaces due to dehydration. Twenty-four sound human premolars and fully developed third molars without periodontitis were selected for the experiment. Roots were separated into 6-7 pieces with an intact root surface area (3×3mm2 ) and then divided into the following groups: fresh group, prolonged dehydration groups (air drying for 1, 2, 4, 12, or 24h), and dehydrated teeth (air drying for 2h) treated with acid (Tris-HCl buffer or citric acid buffer). More than six pieces of root from each group were subjected to scanning electron microscopy (SEM) observation. Captured images were exported to ImageJ software to quantitatively analyze the areas covered with fibers. Statistical significance was determined by comparing the means of the different groups using t-testing or one-way analysis of variance followed by post hoc testing. Fibrous "vegetation" covering the cementum was observed on the fresh root surface by SEM. This was destroyed by dehydration (>1h), resulting in a root surface resembling the "Gobi Desert." The difference was statistically significant (p<.001). Root surface deteriorated by dehydration could be recovered by the re-use of the fibers embedded in the cementum as acid demineralization of the outer layer of cementum exposed the embedded fibers to simulate the geomorphology of fresh root surface. Dehydrated teeth had deteriorated geomorphology of the root surface, which could be reversed by the re-use of the fibers embedded in the cementum using citric acid. Direct evidence from SEM gives new insights into the replantation of dehydrated avulsed teeth.

Comparison between two coating techniques of glass fiber posts treated with hydrogen peroxide 35% on its stability in root canals after cementing using resin cement (In vitro comparative study)

Aim of the Study: This research aims to compare two techniques of coating glass fiber posts to define the best technique that achieves the higher tensile resistance after cementing with dual-cure resin cement into prepared human teeth root canals. Materials and Methods: The sample of this research consisted of 20 extracted human lower premolars which their crowns were cut horizontally 2 mm above the cementoenamel junction, then the root canals were treated endodontically with suitable instruments and divided into 2 groups according to techniques used in coating the glass fiber posts luted into them: Group A: 10 fiber posts were coated with silane before cementing. Group B: 10 fiber posts were coated with unfilled resin before cementing. Each specimen was secured in a universal tensile-test machine. An increasing tensile force was applied on the free part of the fiber post vertically at a crosshead speed of 1 mm/min along with the long axis of the tooth until adhesive failure occurred. Results: The results of the tensile test indicated that there is no statistically significant differences in the average tensile resistance between A and B groups. Conclusions: There was no significant difference in stability strength between the technique of using silane and the technique of using unfilled resin to coat the glass fiber post before cementing with resin cement.

Oxidising agents and its effect on human dentine fluorescence diagnostic measurements.

The ability to determine the effectiveness of root canal disinfection in real-time prior to the final root filling could significantly influence the management and treatment outcomes of root canal therapy. Laser fluorescence devices can detect bacterial fluorescence in real-time, however since quenching of fluorescence is a possible confounding factor, this study explored the effects of oxidising endodontic irrigants on laser fluorescence in dentine. A total of 78 transverse 2 mm thick sections were prepared from the crowns of sterilised extracted non-functional third molars that had been embedded in acrylic resin. The dentine sections where then treated with different concentrations of sodium hypochlorite and hydrogen peroxide, with or without further rinsing with distilled water wash. Fluorescence values were recorded over 24 h using the DIAGNOdent laser fluorescence device. Both sodium hypochlorite and hydrogen peroxide cause quenching of fluorescence readings of human dentine, with reductions of 30-60% in fluorescence readings. The quenching effect occurred quickly, and recovery to baseline readings took from 1 h to 24 h. Rinsing with water reduced but did not eliminate the quenching effect. The use of laser fluorescence to reliably detect root canal bacteria porphyrins in real-time could significantly influence the management of teeth needing root canal therapy. This study highlighted the quenching effects of some root canal irrigants on human teeth root dentine, a variable that should be considered and controlled prior to using lasers to assist in assessing root canal sterility.

Transforaminal and systemic diffusion of an active agent from a zinc oxide eugenol-based endodontic sealer containing hydrocortisone-in an in vivo model.

This study aimed to quantify in vivo the release of hydrocortisone acetate (HCA) contained in a zinc oxide eugenol-based endodontic sealer, in various tissues. Roots of human teeth, shaped with One Shape single file and sealed with Endomethasone N, previously radiolabelled with tritium (3H-HCA), were implanted in the back of 24 mice. Mice were sacrificed at 2, 8, 24, and 48h to evaluate and quantify the amount of radioactivity in subcutaneous tissues surrounding the apex (periapical-like) of the implanted teeth, blood, spleen, kidneys, liver, and urine. Radioactivity was released from the apex of the tooth into the periapical-like tissues with a peak measured at 2h post-implantation (2.25% of the initial radioactivity/g). This quantity decreased significantly over time between 2h and each time points. Radioactivity was still measured up to 48h in the periapical-like tissues (0.42% of the initial radioactivity). The same pattern of kinetic was observed for all organs. The total quantity of radioactivity significantly decreased over time from 4.36% measured 2h post-implantation to 0.74% at 48h. Finally, about 10% of the initial radioactivity from Endomethasone N used to fill the root canal was retrieved after 48h in the urine. This study demonstrated that radioactive-HCA from Endomethasone N can diffuse through the apex of the root canal and follow a classical pharmacokinetics. This mouse model shows that radioactive-HCA can diffuse through the apex and do not accumulate in periapical-like tissues and organs.

Irradiation with blue light-emitting diode enhances osteogenic differentiation of stem cells from the apical papilla.

This study aimed to evaluate the effects of low-energy blue LED irradiation on the osteogenic differentiation of stem cells from the apical papilla (SCAPs). SCAPs were derived from human tooth root tips and were irradiated with 0 (control group), 1J/cm2, 2J/cm2, 3J/cm2, or 4J/cm2 blue light in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP), alizarin red staining, and real-time polymerase chain reaction (RT-PCR). The results of the MTT assay indicated that SCAPs in the LED groups exhibited a lower proliferation rate than those in the control group, and there were statistically differences between the 2J/cm2, 3J/cm2, and 4J/cm2 groups and the control group (P < 0.05). The results of the ALP and alizarin red analyses showed that blue LED promoted osteogenic differentiation of the SCAPs. And 4J/cm2 blue light upregulates the expression levels of the osteogenic/dentinogenic genes ALP, dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), and osteocalcin (OCN) in SCAPs. Our results confirmed that low-energy blue LED at 1J/cm2, 2J/cm2, 3J/cm2, and 4J/cm2 could inhibit the proliferation of SCAPs and promotes osteogenic differentiation of SCAPs. Further in vitro studies are required to explore the mechanisms of the effects by low-energy blue LED.

Comparative evaluation of twisted and protaper files in pediatric endodontics – In-vitro study

Aim: The aim of the present in-vitro study was to evaluate the cutting efficiency of two nickel-titanium (NiTi) file systems, Twisted File (TF) and ProTaper file. Materials and Methods: Thirty extracted human primary tooth root canals were randomly divided into two groups with 15 root canals each. All the root canals after injected with Indian ink were instrumented with ProTaper Rotary NiTi files and Rotary NiTi TFs. All the root canals were then cleared to make them transparent. After the teeth appeared clear, they were observed under a stereomicroscope to check for residual Indian ink in the canals and scored. The data thus obtained were statistically analyzed with Chi-square test and Mann–Whitney test. Results: The mean of scoring of Group I, i.e., Rotary TF system, was 1.0. The mean of scoring of Group II, i.e., ProTaper Rotary file system, was 1.40. Conclusions: Root canal instrumentation of primary teeth showed nonsignificant differences in the cutting ability between the two systems of ProTaper and TF Rotary which were investigated, but signified a more even and uniform removal of dentin with the TF system.

Dental pulp stem cells overexpressing stromal-derived factor-1α and vascular endothelial growth factor in dental pulp regeneration.

The current study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) overexpressing dental pulp stem cells (DPSCs) in vascularized dental pulp regeneration in vivo. Human DPSCs were transfected with VEGF or SDF-1α using premade lentiviral particles. Overexpression was verified by quantitative polymerase chain reaction (q-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot analysis. Effects of SDF-1α and VEGF overexpressing DPSCs on their proliferation (CCK-8 and MTT assays) and endothelial vascular-tube formation (Matrigel assay) were investigated in vitro. Human tooth roots sectioned into 6-mm segments were injected with gene-modified DPSCs encapsulated in PuraMatrix hydrogel and implanted in the dorsum of severe-combined-immunodeficient (SCID) mice. Implants were retrieved after 4weeks and examined for regenerated pulp-like tissue and vascularization using histology and immunohistochemistry. p < 0.05 was considered statistically significant. Gene-modified DPSCs expressed significantly high levels (p < 0.05) of SDF-1α and VEGF mRNA and proteins, respectively. Transfected DPSCs showed a significantly higher cell proliferation compared to that of wild-type DPSCs. Furthermore, they enhanced endothelial cell migration and vascular-tube formation on Matrigel in vitro. When injected into tooth root canals and implanted in vivo, DPSCs/SDF-1α + DPSCs/VEGF-mixed group resulted in significantly increased length of regenerated pulp-like tissue within the root canals compared to that of wild-type DPSCs/VEGF and DPSCs/SDF-1α groups. Vessel area density was significantly higher in DPSCs/SDF-1α and mixed DPSCs/SDF-1α + DPSCs/VEGF groups than in DPSCs-VEGF alone or wild-type DPSCs groups. A combination of VEGF-overexpressing and SDF-1α-overexpressing DPSCs could enhance the area of vascularized dental pulp regeneration in vivo. Enhancing vascularization in pulp regeneration is crucial to overcome the clinical limitation of the limited blood supply to the root canals via a small apical foramen enclosed by hard dentin.

Imaging of growth factors on a human tooth root canal by surface-enhanced Raman spectroscopy.

Endodontic treatment of immature permanent teeth with necrotic pulp poses several clinical challenges and is one of the most demanding interventions in endodontics. Recently, with new discoveries in the field of tissue engineering, novel treatment protocols have been established. The most promising treatment modality is revascularization, whose integral part is the exposure of collagen matrix and embedded growth factors. However, optimization of the treatment protocol requires a development of analytical procedures able to analyze growth factors directly on the sample surface. In this work, method based on surface-enhanced Raman spectroscopy (SERS) was developed to investigate the influence of the time of the medical treatment using EDTA on exposure and accessibility of the growth factors, namely TGF-ß1, BMP-2, and bFGF on the dentine surface. The nanotags, which consist of magnetic Fe3O4@Ag nanocomposite covalently functionalized by tagged antibodies (anti-TGF-ß1-Cy3, anti-BMP-2-Cy5, and anti-bFGF-Cy7), were employed as a SERS substrate. Each antibody was coupled with a unique label allowing us to perform a parallel analysis of all three growth factors within one analytical run. Developed methodology presents an interesting alternative to a fluorescence microscopy and in contrary allows evaluating a chemical composition and thus minimizing possible false-positive results. Graphical abstract.

Pulp Regeneration by 3-dimensional Dental Pulp Stem Cell Constructs

Dental pulp regeneration therapy for the pulpless tooth has attracted recent attention, and clinical trial studies are underway with the tissue engineering approach. However, there remain many concerns, including the extended period for regenerating the dental pulp. In addition, the use of scaffolds increases the risk of inflammation and infection. To establish a basic technology for novel dental pulp regenerative therapy that allows transplant of pulp-like tissue, we attempted to fabricate scaffold-free 3-dimensional (3D) cell constructs composed of dental pulp stem cells (DPSCs). Furthermore, we assessed viability of these 3D DPSC constructs for dental pulp regeneration through in vitro and in vivo studies. For the in vitro study, we obtained 3D DPSC constructs by shaping sheet-like aggregates of DPSCs with a thermoresponsive hydrogel. DPSCs within constructs remained viable even after prolonged culture; furthermore, 3D DPSC constructs possessed a self-organization ability necessary to serve as a transplant tissue. For the in vivo study, we filled the human tooth root canal with DPSC constructs and implanted it subcutaneously into immunodeficient mice. We found that pulp-like tissues with rich blood vessels were formed within the human root canal 6 wk after implantation. Histologic analyses revealed that transplanted DPSCs differentiated into odontoblast-like mineralizing cells at sites in contact with dentin; furthermore, human CD31–positive endothelial cells were found at the center of regenerated tissue. Thus, the self-organizing ability of 3D DPSC constructs was active within the pulpless root canal in vivo. In addition, blood vessel–rich pulp-like tissues can be formed with DPSCs without requiring scaffolds or growth factors. The technology established in this study allows us to prepare DPSC constructs with variable sizes and shapes; therefore, transplantation of DPSC constructs shows promise for regeneration of pulpal tissue in the pulpless tooth.

Global variation in human tooth crown and root morphology - anthropological and forensic applications - 1

Global variation in human tooth crown and root morphology - anthropological and forensic applications - 1

Global variation in human tooth crown and root morphology - anthropological and forensic applications - 2

Global variation in human tooth crown and root morphology - anthropological and forensic applications - 2

Comparison of Commercial Calcium Hydroxide Pastes for Prolonged Antibacterial Effect using a Colourimetric Assessment.

The anti-microbial activity of calcium hydroxide pastes used in endodontics is dependent on establishing high levels of hydroxyl ions in dentine. This study investigated hydroxyl ion diffusion from different commercial calcium hydroxide pastes using a novel colourimetric method. In this method, human tooth roots were stained with anthocyanin dye, which changed their colour according to the local pH conditions. Prepared root canals were filled with pastes formulated with the vehicle of water (Pulpdent™, Calasept Plus™), polyethylene glycol (PEG) (Calmix™) or a mixture of water, PEG and ibuprofen (Odontocide™). The changes in dye colour at fixed distances from the canal wall were monitored using standardised digital photography over a period of 3 weeks. A repeated measures analysis tracked changes in each root from baseline. Release of hydroxyl ions varied between the different commercial compositions containing water or PEG as solvents. The colour changes in the dentine, due to released hydroxyl ions, were greatest and more prolonged for completely non-aqueous compositions, when using PEG 400 as the vehicle. When water was present in the product, the duration of the pH changes was shorter. This was attributed to the presence of hydroxyl ions in the water (the common-ion effect) and a more vigorous buffering of hydroxyl ions by dentine proteins.

Book Review: Human Tooth Crown and Root Morphology and Dental Morphology for Anthropology: An Illustrated Manual

Book Review: Human Tooth Crown and Root Morphology and Dental Morphology for Anthropology: An Illustrated Manual

In vitro performance of DIAGNOdent laser fluorescence device for dental calculus detection on human tooth root surfaces.

ObjectiveThis study assessed the reproducibility of a red diode laser device, and its capability to detect dental calculus in vitro on human tooth root surfaces.Material and methodsOn each of 50 extracted teeth, a calculus-positive and calculus-free root surface was evaluated by two independent examiners with a low-power indium gallium arsenide phosphide diode laser (DIAGNOdent) fitted with a periodontal probe-like sapphire tip and emitting visible red light at 655 nm wavelength. Laser autofluorescence intensity readings of examined root surfaces were scored on a 0–99 scale, with duplicate assessments performed using the laser probe tip directed both perpendicular and parallel to evaluated tooth root surfaces. Pearson correlation coefficients of untransformed measurements, and kappa analysis of data dichotomized with a >40 autofluorescence intensity threshold, were calculated to assess intra- and inter-examiner reproducibility of the laser device. Mean autofluorescence intensity scores of calculus-positive and calculus-free root surfaces were evaluated with the Student’s t-test.ResultsExcellent intra- and inter-examiner reproducibility was found for DIAGNOdent laser autofluorescence intensity measurements, with Pearson correlation coefficients above 94%, and kappa values ranging between 0.96 and 1.0, for duplicate readings taken with both laser probe tip orientations. Significantly higher autofluorescence intensity values were measured when the laser probe tip was directed perpendicular, rather than parallel, to tooth root surfaces. However, calculus-positive roots, particularly with calculus in markedly-raised ledges, yielded significantly greater mean DIAGNOdent laser autofluorescence intensity scores than calculus-free surfaces, regardless of probe tip orientation. DIAGNOdent autofluorescence intensity values >40 exhibited a stronger association with calculus (36.6 odds ratio) then measurements of ≥5 (20.1 odds ratio) when the laser probe tip was advanced parallel to root surfaces.ConclusionsExcellent intra- and inter-examiner reproducibility of autofluorescence intensity measurements was obtained with the DIAGNOdent laser fluorescence device on human tooth roots. Calculus-positive root surfaces exhibited significantly greater DIAGNOdent laser autofluorescence than calculus-free tooth roots, even with the laser probe tip directed parallel to root surfaces. These findings provide further in vitro validation of the potential utility of a DIAGNOdent laser fluorescence device for identifying dental calculus on human tooth root surfaces.

Suitability of Different Natural and Synthetic Biomaterials for Dental Pulp Tissue Engineering.

Dental pulp tissue engineering is possible after insertion of pulpal stem cells combined with a scaffold into empty root canals. Commonly used biomaterials are collagen or poly(lactic) acid, which are either difficult to modify or to insert into such a narrow space. New hydrogel scaffolds with bioactive, specifically tailored functions could optimize the conditions for this approach. Different synthetic and natural hydrogels were tested for their suitability to engineer dental pulp. Two functionalized modifications of polyethylene glycol were developed in this study and compared to a self-assembling peptide, as well as to collagen and fibrin. Cell viability of dental pulp stem cells in test materials was assessed over two weeks. Cells in selected test materials laden with dentin-derived growth factors were inserted into human tooth roots and implanted subcutaneously into immunocompromised mice. In vitro cell culture exhibited distinct differences between scaffold types, where viability was significantly higher in natural compared to synthetic materials. In vivo experiments showed considerable differences regarding scaffold degradation, soft tissue formation, vascularization, and odontoblast-like cell differentiation. Fibrin appeared most suitable to enable generation of a pulp-like tissue and differentiation of cells into odontoblasts at the cell-dentin interface. In conclusion, natural materials, especially fibrin, proved to be superior compared to synthetic scaffolds regarding cell viability and dental pulp-like tissue formation.

Periodontal ligament stem/progenitor cells with protein-releasing scaffolds for cementum formation and integration on dentin surface

ABSTRACTPurpose/Aim: Cementogenesis is a critical step in periodontal tissue regeneration given the essential role of cementum in anchoring teeth to the alveolar bone. This study is designed to achieve integrated cementum formation on the root surfaces of human teeth using growth factor–releasing scaffolds with periodontal ligament stem/progenitor cells (PDLSCs). Materials and methods: Human PDLSCs were sorted by CD146 expression, and characterized using CFU-F assay and induced multi-lineage differentiation. Polycaprolactone scaffolds were fabricated using 3D printing, embedded with poly(lactic-co-glycolic acids) (PLGA) microspheres encapsulating connective tissue growth factor (CTGF), bone morphogenetic protein-2 (BMP-2), or bone morphogenetic protein-7 (BMP-7). After removing cementum on human tooth roots, PDLSC-seeded scaffolds were placed on the exposed dentin surface. After 6-week culture with cementogenic/osteogenic medium, cementum formation and integration were evaluated by histomorphometric analysis, immunofluorescence, and qRT-PCR. Results: Periodontal ligament (PDL) cells sorted by CD146 and single-cell clones show a superior clonogenecity and multipotency as compared with heterogeneous populations. After 6 weeks, all the growth factor–delivered groups showed resurfacing of dentin with a newly formed cementum-like layer as compared with control. BMP-2 and BMP-7 showed de novo formation of tissue layers significantly thicker than all the other groups, whereas CTGF and BMP-7 resulted in significantly improved integration on the dentin surface. The de novo mineralized tissue layer seen in BMP-7-treated samples expressed cementum matrix protein 1 (CEMP1). Consistently, BMP-7 showed a significant increase in CEMP1 mRNA expression. Conclusion: Our findings represent important progress in stem cell–based cementum regeneration as an essential part of periodontium regeneration.

The effect of age on Ca, Cl, K, Mg, Mn, Na, P and Sr contents in roots of human female permanent teeth

The effect of age on Ca, Cl, K, Mg, Mn, Na, P, and Sr contents in intact roots of permanent teeth of 38 apparently healthy women aged 15–55 years was investigated by neutron activation analysis. It was found that mass fractions of Ca, Cl, K, Mg, and P are at a maximum in the age range 25–35 years and above 35 years there is a monotonic decrease. A statistically significant age-dependent decrease was shown only for Mg and P. The Sr content significantly increases with age. The mass fractions of Mn and Na remain stable for the ages 15–55 years.

Pulp regeneration in a full-length human tooth root using a hierarchical nanofibrous microsphere system

While pulp regeneration using tissue engineering strategy has been explored for over a decade, successful regeneration of pulp tissues in a full-length human root with a one-end seal that truly simulates clinical endodontic treatment has not been achieved. To address this challenge, we designed and synthesized a unique hierarchical growth factor-loaded nanofibrous microsphere scaffolding system. In this system, vascular endothelial growth factor (VEGF) binds with heparin and is encapsulated in heparin-conjugated gelatin nanospheres, which are further immobilized in the nanofibers of an injectable poly(l-lactic acid) (PLLA) microsphere. This hierarchical microsphere system not only protects the VEGF from denaturation and degradation, but also provides excellent control of its sustained release. In addition, the nanofibrous PLLA microsphere integrates the extracellular matrix-mimicking architecture with a highly porous injectable form, efficiently accommodating dental pulp stem cells (DPSCs) and supporting their proliferation and pulp tissue formation. Our in vivo study showed the successful regeneration of pulp-like tissues that fulfilled the entire apical and middle thirds and reached the coronal third of the full-length root canal. In addition, a large number of blood vessels were regenerated throughout the canal. For the first time, our work demonstrates the success of pulp tissue regeneration in a full-length root canal, making it a significant step toward regenerative endodontics. Statement of SignificanceThe regeneration of pulp tissues in a full-length tooth root canal has been one of the greatest challenges in the field of regenerative endodontics, and one of the biggest barriers for its clinical application. In this study, we developed a unique approach to tackle this challenge, and for the first time, we successfully regenerated living pulp tissues in a full-length root canal, making it a significant step toward regenerative endodontics.This study will make positive scientific impact and interest the broad and multidisciplinary readership in the dental biomaterials and craniofacial tissue engineering community.

A High-resolution Immunohistochemical Method for studying Receptor Expression on the Periodontal Ligament of Whole-mount Human Tooth Roots

ABSTRACTAimsOur laboratory has found that lysophosphatidic acid (LPA) and its cognate receptors [LPARs, (LPA1–6)] expressed by human gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) play key roles in oral fibroblast homeostasis and are implicated in the inflammation seen in periodontal disease. We have reported that PDLF express LPA1 and LPA3; however, information on the gross topographic distribution of LPARs in the periodontal ligament (PDL) was lacking, and therefore, we developed a simple method forin situlabeling of LPARs in the PDL of extracted teeth.Materials and methodsSectioning or grinding thin sections of demineralized or native teeth and periodontium have long been the standard methodologies used to assess biomarker distribution in the PDL; however, we modified traditional immunohistochemical labeling and used whole teeth with fixed, solvent permeabilized PDLs.ResultsLPA1 and LPA3 were specifically labeled in the PDL and could be visualized at both the macroand micro-levels.ConclusionThis technique effectively labeled LPARs, and it can serve as a basis for thein situvisualization of other biomolecules expressed in the PDL.Clinical SignificanceThe ability to observe PDL LPAR distribution at the macro-level complements the microscopic data, and it is useful for detecting and documenting molecular changes in the PDL/PDLF that were brought about by age, experimental treatments, or pathologies like periodontal disease.How to cite this articleCerutis DR, Headen KV, Ogunleye AO, Williams DE. A High-resolution Immunohistochemical Method for studying Receptor Expression on the Periodontal Ligament of Whole-mount Human Tooth Roots. Int J Experiment Dent Sci 2016;5(2):99-103.

β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine derivatives eradicate C. albicans in an ex vivo human dentinal tubule model by inhibiting sterol 14-α demethylase and cAMP pathway

BackgroundFurther quest for new anti-fungal compounds with proven mechanisms of action arises due to resistance and dose limiting toxicity of existing agents. Among the human fungal pathogens C. albicans predominate by infecting several sites in the body and in particular oral cavity and root canals of human tooth. MethodsIn the present study, we screened a library of β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine compounds against Candida sp. Detailed molecular studies were carried out with the active compound 3 on C. albicans. Morphological damage and antibiofilm activity of compound 3 on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. In silico docking studies were carried out to elucidate the mechanism of action of compound 3. Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model. ResultsScreening data showed that several new compounds were active against Candida sp. Among them, Compound 3 was most potent and exerted time kill effect at 4h, post antifungal effect up to 6h. When used in combination with fluconazole or nystatin, compound 3 revealed an minimum inhibitory concentration (MIC) decrease by 4 fold for both drugs used. In-depth molecular studies with compound 3 on C. albicans showed that this compound inhibited yeast to hyphae (Y-H) conversion and this involved the cAMP pathway. Further, SEM images of C. albicans showed that compound 3 caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, in silico studies revealed that compound 3 docks with the active site of the key enzyme 14-α-demethylase and this might inhibit ergosterol synthesis. In support of this, ergosterol levels were found to be decreased by 32 fold in compound 3 treated samples as analyzed by high performance liquid chromatography (HPLC). Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed 83% eradication of C. albicans and a 6 log reduction in colony forming unit (CFU) after 24h treatment in the infected tooth samples in this model. ConclusionCompound 3 was found to be very effective in eradicating C. albicans by inhibiting cAMP pathway and ergosterol biosynthesis. General SignificanceThe results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.