Effects of ciprofloxacin-containing antimicrobial scaffolds on dental pulp stem cell viability-In vitro studies

OBJECTIVE: A combination of antibiotics, including but not limited to metronidazole (MET) and ciprofloxacin (CIP), has been indicated to eradicate bacteria in necrotic immature permanent teeth prior to regenerative procedures. It has been shown clinically that antibiotic pastes may lead to substantial stem cell death. The aim of this study was to synthesise scaffolds containing various concentrations of CIP to enhance cell viability while preserving antimicrobial properties. DESIGN: Polydioxanone (PDS)-based electrospun scaffolds were processed with decreasing CIP concentrations (25-1 wt.%) and morphologically evaluated using scanning electron microscopy (SEM). Cytotoxicity assays were performed to determine whether the amount of CIP released from the scaffolds would lead to human dental pulp stem cell (hDPSC) toxicity. Similarly, WST-1 assays were performed to evaluate the impact of CIP release on hDPSC proliferation. Pure PDS scaffolds and saturated double antibiotic solution MET/CIP (DAP) served as both positive and negative controls, respectively. Antibacterial efficacy against E. faecalis (Ef) was tested. RESULTS: A significant decrease in hDPSC' viability at concentrations 5-25 wt.% was observed. However, concentrations below 5wt.% did not impair cell viability. Data from the WST-1 assays indicated no detrimental impact on cell proliferation for scaffolds containing 2.5 wt.% CIP or less. Significant antimicrobial properties were seen for CIP-scaffolds at lower concentrations (i.e., 1 and 2.5 wt.%). CONCLUSION: The obtained data demonstrated that a reduced concentration of CIP incorporated into PDS-based scaffolds maintains its antimicrobial properties while enhancing viability and proliferation of dental pulp stem cells

Advanced Scaffolds for Dental Pulp and Periodontal Regeneration

No current therapy promotes root canal disinfection and regeneration of the pulp-dentin complex in cases of pulp necrosis. Antibiotic pastes used to eradicate canal infection negatively affect stem cell survival. Three-dimensional easy-to-fit antibiotic-eluting nanofibers, combined with injectable scaffolds, enriched or not with stem cells and/or growth factors, may increase the likelihood of achieving predictable dental pulp regeneration. Periodontitis is an aggressive disease that impairs the integrity of tooth-supporting structures and may lead to tooth loss. The latest advances in membrane biomodification to endow needed functionalities and technologies to engineer patient-specific membranes/constructs to amplify periodontal regeneration are presented

A novel patient‐specific three‐dimensional drug delivery construct for regenerative endodontics

Evoked bleeding (EB) clinical procedure, comprising a disinfection step followed by periapical tissue laceration to induce the ingrowth of undifferentiated stem cells from the periodontal ligament and alveolar bone, is currently the only regenerative‐based therapeutic approach to treating pulp tissue necrosis in undeveloped (immature) permanent teeth approved in the United States. Yet, the disinfection step using antibiotic‐based pastes leads to cytotoxic, warranting a biocompatible strategy to promote root canal disinfection with no or minimal side‐effects to maximize the regenerative outcomes. The purpose of this investigation was to develop a tubular three‐dimensional (3D) triple antibiotic‐eluting construct for intracanal drug delivery. Morphological (scanning electron microscopy), chemical (Fourier transform infrared spectroscopy), and mechanical (tensile testing) characteristics of the polydioxanone‐based triple antibiotic‐eluting fibers were assessed. The antimicrobial properties of the tubular 3D constructs were determined in vitro and in vivo using an infected (Actinomyces naeslundii) dentin tooth slice model and a canine method of periapical disease, respectively. The in vitro data indicated significant antimicrobial activity and the ability to eliminate bacterial biofilm inside dentinal tubules. In vivo histological findings demonstrated that, using the EB procedure, the tubular 3D triple antibiotic‐eluting construct allowed the formation of an appropriate environment that led to apex closure and the ingrowth of a thin layer of osteodentin‐like tissue into the root canal. Taken together, these findings indicate that our novel drug delivery construct is a promising biocompatible disinfection strategy for immature permanent teeth with necrotic pulps. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1576–1586, 2019.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149514/1/jbmb34250_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149514/2/jbmb34250.pd

Injectable Highly Tunable Oligomeric Collagen Matrices for Dental Tissue Regeneration

Current stem cell transplantation approaches lack efficacy, because they limit cell survival and retention and, more importantly, lack a suitable cellular niche to modulate lineage-specific differentiation. Here, we evaluate the intrinsic ability of type I oligomeric collagen matrices to modulate dental pulp stem cells (DPSCs) endothelial and odontogenic differentiation as a potential stem cell-based therapy for regenerative endodontics. DPSCs were encapsulated in low-stiffness (235 Pa) and high-stiffness (800 Pa) oligomeric collagen matrices and then evaluated for long-term cell survival, as well as endothelial and odontogenic differentiation following in vitro cell culture. Moreover, the effect of growth factor incorporation, i.e., vascular endothelial growth factor (VEGF) into 235 Pa oligomeric collagen or bone morphogenetic protein (BMP2) into the 800 Pa oligomeric collagen counterpart on endothelial or odontogenic differentiation of encapsulated DPSCs was investigated. DPSCs-laden oligomeric collagen matrices allowed long-term cell survival. Real time polymerase chain reaction (RT-PCR) data showed that the DPSCs cultured in 235 Pa matrices demonstrated an increased expression of endothelial markers after 28 days, and the effect was enhanced upon VEGF incorporation. There was a significant increase in alkaline phosphatase (ALP) activity at Day 14 in the 800 Pa DPSCs-laden oligomeric collagen matrices, regardless of BMP2 incorporation. However, Alizarin S data demonstrated higher mineralization by Day 21 and the effect was amplified in BMP2-modified matrices. Herein, we present key data that strongly support future research aimed at clinical translation of an injectable oligomeric collagen system for delivery and fate regulation of DPSCs to enable pulp and dentin regeneration at specific locations of the root canal system

Endothelial-derived interleukin-6 induces cancer stem cell motility by generating a chemotactic gradient towards blood vessels

Recent evidence suggests that the metastatic spread of head and neck squamous cell carcinomas (HNSCC) requires the function of cancer stem cells endowed with multipotency, self-renewal, and high tumorigenic potential. We demonstrated that cancer stem cells reside in perivascular niches and are characterized by high aldehyde dehydrogenase (ALDH) activity and high CD44 expression (ALDHhighCD44high) in HNSCC. Here, we hypothesize that endothelial cell-secreted interleukin-6 (IL-6) contributes to tumor progression by enhancing the migratory phenotype and survival of cancer stem cells. Analysis of tissue microarrays generated from the invasive fronts of 77 HNSCC patients followed-up for up to 11 years revealed that high expression of IL-6 receptor (IL-6R) (p=0.0217) or co-receptor gp130 (p=0.0422) correlates with low HNSCC patient survival. We observed that endothelial cell-secreted factors induce epithelial to mesenchymal transition (EMT) and enhance invasive capacity of HNSCC cancer stem cells. Conditioned medium from CRISPR/Cas9-mediated IL-6 knockout primary human endothelial cells is less chemotactic for cancer stem cells in a microfluidics-based system than medium from control endothelial cells (p < 0.05). Blockade of the IL-6 pathway with a humanized anti-IL-6R antibody (tocilizumab) inhibited endothelial cell-induced motility in vitro and decreased the fraction of cancer stem cells in vivo. Notably, xenograft HNSCC tumors vascularized with IL-6-knockout endothelial cells exhibited slower tumor growth and smaller cancer stem cell fraction. These findings demonstrate that endothelial cell-secreted IL-6 enhances the motility and survival of highly tumorigenic cancer stem cells, suggesting that endothelial cells can create a chemotactic gradient that enables the movement of carcinoma cells towards blood vessels

Periodontal disease affects oral cancer progression in a surrogate animal model for tobacco exposure

For decades, the link between poor oral hygiene and the increased prevalence of oral cancer has been suggested. Most recently, emerging evidence has suggested that chronic inflammatory diseases from the oral cavity (e.g., periodontal disease), to some extent, play a role in the development of oral squamous cell carcinoma (OSCC). The present study aimed to explore the direct impact of biofilm-induced periodontitis in the carcinogenesis process using a tobacco surrogate animal model for oral cancer. A total of 42 Wistar rats were distributed into four experimental groups: Control group, periodontitis (Perio) group, 4-nitroquinoline 1-oxide (4-NQO) group and 4NQO/Perio group. Periodontitis was stimulated by placing a ligature subgingivally, while oral carcinogenesis was induced by systemic administration of 4NQO in the drinking water for 20 weeks. It was observed that the Perio, 4NQO and 4NQO/Perio groups presented with significantly higher alveolar bone loss compared with that in the control group. Furthermore, all groups receiving 4NQO developed lesions on the dorsal surface of the tongue; however, the 4NQO/Perio group presented larger lesions compared with the 4NQO group. There was also a modest overall increase in the number of epithelial dysplasia and OSCC lesions in the 4NQO/Perio group. Notably, abnormal focal activation of cellular differentiation (cytokeratin 10-positive cells) that extended near the basal cell layer of the mucosa was observed in rats receiving 4NQO alone, but was absent in rats receiving 4NQO and presenting with periodontal disease. Altogether, the presence of periodontitis combined with 4NQO administration augmented tumor size in the current rat model and tampered with the protective mechanisms of the cellular differentiation of epithelial cells

Dental pulp tissue engineering

Dental pulp is a highly specialized mesenchymal tissue, which have a restrict regeneration capacity due to anatomical arrangement and post-mitotic nature of odontoblastic cells. Entire pulp amputation followed by pulp-space disinfection and filling with an artificial material cause loss of a significant amount of dentin leaving as life-lasting sequelae a non-vital and weakened tooth. However, regenerative endodontics is an emerging field of modern tissue engineering that demonstrated promising results using stem cells associated with scaffolds and responsive molecules. Thereby, this article will review the most recent endeavors to regenerate pulp tissue based on tissue engineering principles and providing insightful information to readers about the different aspects enrolled in tissue engineering. Here, we speculate that the search for the ideal combination of cells, scaffolds, and morphogenic factors for dental pulp tissue engineering may be extended over future years and result in significant advances in other areas of dental and craniofacial research. The finds collected in our review showed that we are now at a stage in which engineering a complex tissue, such as the dental pulp, is no longer an unachievable and the next decade will certainly be an exciting time for dental and craniofacial research

BDNF/TrkB Signaling as a Potential Novel Target in Pediatric Brain Tumors: Anticancer Activity of Selective TrkB Inhibition in Medulloblastoma Cells

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Deregulation of brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) signaling has been associated with increased proliferative capabilities, invasiveness, and chemoresistance in several types of cancer. However, the relevance of this pathway in MB remains unknown. Here, we show that the selective TrkB inhibitor N-[2-[[(hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide (ANA-12) markedly reduced the viability and survival of human cell lines representative of different MB molecular subgroups. These findings provide the first evidence supporting further investigation of TrkB inhibition as a potential novel strategy for MB treatment

Hyaluronic acid hydrogels incorporating platelet lysate enhance human pulp cell proliferation and differentiation

The restoration of dentine-pulp complex remains a challenge for dentists; nonetheless, it has been poorly addressed. An ideal system should modulate the host response, as well as enable the recruitment, proliferation and differentiation of relevant progenitor cells. Herein was proposed a photocrosslinkable hydrogel system based on hyaluronic acid (HA) and platelet lysate (PL). PL is a cocktail of growth factors (GFs) and cytokines involved in wound healing orchestration, obtained by the cryogenic processing of platelet concentrates, and was expected to provide the HA hydrogels specific biochemical cues to enhance pulp cellsâ recruitment, proliferation and differentiation. Stable HA hydrogels incorporating PL (HAPL) were prepared after photocrosslinking of methacrylated HA (Met-HA) previously dissolved in PL, triggered by the Ultra Violet activated photoinitiator Irgacure 2959. Both the HAPL and plain HA hydrogels were shown to be able to recruit cells from a cell monolayer of human dental pulp stem cells (hDPSCs) isolated from permanent teeth. The hDPCs were also seeded directly over the hydrogels (5 Ã 104 cells/hydrogel) and cultured in osteogenic conditions. Cell metabolism and DNA quantification were higher, in all time-points, for PL supplemented hydrogels (p < 0,05). Alkaline phosphatase (ALPL) activity and calcium quantification peaks were observed for the HAPL group at 21 days (p < 0,05). The gene expression for ALPL and COLIA1 was up-regulated at 21 days to HAPL, compared with HA group (p < 0,05). Within the same time point, the gene expression for RUNX2 did not differ between the groups. Overall, data demonstrated that the HA hydrogels incorporating PL increased the cellular metabolism and stimulate the mineralized matrix deposition by hDPSCs, providing clear evidence of the potential of the proposed system for the repair of damaged pulp/dentin tissue and endodontics regeneration.LFDA acknowledges Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the grant 2014/12017-8. Portuguese Foundation for Science and Technology (FCT) for PSB PhD grant SFRH/BD/73403/2010, MTR post-doctoral grant (SFRH/BPD/111729/2015), MEG grant (IF/00685/2012), and RECOGNIZE project (UTAP-ICDT/CTM-BIO/0023/2014), RL3-TECT - NORTE-07-0124-FEDER-000020 project co-financed by ON.2 (NSRF) through ERD. This study also received financial support from FCT/Ministério da Ciência, Tecnologia, e Ensino Superior (FCT/MCTES) and Fundo Social Europeu through Programa Operacional do Capital Humano (FSE/POCH) PD/59/2013 for the LA ICVS-3Bs (UID/Multi/50026/2013). The authors would like to thank Maurizio Gulino, for its support in the in vitro experiments and Maló Clinic, Porto, Dra Ana Ferro and Dr Bruno Queridinha for the donation of permanent teethinfo:eu-repo/semantics/publishedVersio

Expression and pharmacological inhibition of TrkB and EGFR in glioblastoma

A member of the Trk family of neurotrophin receptors, tropomyosin receptor kinase B (TrkB, encoded by the NTRK2 gene) is an increasingly important target in various cancer types, including glioblastoma (GBM). EGFR is among the most frequently altered oncogenes in GBM, and EGFR inhibition has been tested as an experimental therapy. Functional interactions between EGFR and TrkB have been demonstrated. In the present study, we investigated the role of TrkB and EGFR, and their interactions, in GBM. Analyses of NTRK2 and EGFR gene expression from The Cancer Genome Atlas (TCGA) datasets showed an increase in NTRK2 expression in the proneural subtype of GBM, and a strong correlation between NTRK2 and EGFR expression in glioma CpG island methylator phenotype (G-CIMP+) samples. We showed that when TrkB and EGFR inhibitors were combined, the inhibitory effect on A172 human GBM cells was more pronounced than when either inhibitor was given alone. When U87MG GBM cells were xenografted into the flank of nude mice, tumor growth was delayed by treatment with TrkB and EGFR inhibitors, given alone or combined, only at specific time points. Intracranial GBM growth in mice was not significantly affected by drug treatments. Our findings indicate that correlations between NTRK2 and EGFR expression occur in specific GBM subgroups. Also, our results using cultured cells suggest for the first time the potential of combining TrkB and EGFR inhibition for the treatment of GBM