Athina Bakopoulou

Comparative analysis of in vitro osteo/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP).

OBJECTIVE The aim of this study was to compare the in vitro osteo/odontogenic differentiation potential of mesenchymal stem cells (MSCs) derived from the dental pulp (dental pulp stem cells - DPSCs) or the apical papilla (stem cells from the apical papilla - SCAP) of permanent developing teeth. DESIGN DPSCs and SCAP cultures were established from impacted third molars of young healthy donors at the stage of root development. Cultures were analysed for stem cell markers, including STRO-1, CD146, CD34 and CD45 using flow cytometry. Cells were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4) and β-glycerophosphate. Cultures were analysed for morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers (dentine sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN, alkaline phosphatase-ALP), using immunocytochemistry and reverse transcriptase-polymerase chain reaction. RESULTS All DPSCs and SCAP cultures were positive for STRO-1, CD146 and CD34, in percentages varying according to cell type and donor, but negative for CD45. Both types of MSCs displayed an active potential for cellular migration, organization and mineralization, producing 3D mineralized structures. These structures progressively expressed differentiation markers, including DSPP, BSP, OCN, ALP, having the characteristics of osteodentin. SCAP, however, showed a significantly higher proliferation rate and mineralization potential, which might be of significance for their use in bone/dental tissue engineering. CONCLUSIONS This study provides evidence that different types of dental MSCs can be used in tissue engineering/regeneration protocols as an approachable stem cell source for osteo/odontogenic differentiation and biomineralization that could be further applied for stem cell-based clinical therapies.

Molecular Toxicology of Substances Released from Resin-Based Dental Restorative Materials

Resin-based dental restorative materials are extensively used today in dentistry. However, significant concerns still remain regarding their biocompatibility. For this reason, significant scientific effort has been focused on the determination of the molecular toxicology of substances released by these biomaterials, using several tools for risk assessment, including exposure assessment, hazard identification and dose-response analysis. These studies have shown that substances released by these materials can cause significant cytotoxic and genotoxic effects, leading to irreversible disturbance of basic cellular functions. The aim of this article is to review current knowledge related to dental composites’ molecular toxicology and to give implications for possible improvements concerning their biocompatibility.

Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth

OBJECTIVES The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells. METHODS Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH(2)PO(4),β-glycerophosphate and L-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5mM) and TEGDMA (0.05-0.25mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2mM) and TEGDMA (1mM) were also evaluated. RESULTS DTSCs cultures were positive for STRO-1 (7.53±2.5%), CD146 (91.79±5.41%), CD34 (11.87±3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated. SIGNIFICANCE These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.

New Ti-Alloys and Surface Modifications to Improve the Mechanical Properties and the Biological Response to Orthopedic and Dental Implants: A Review

Titanium implants are widely used in the orthopedic and dentistry fields for many decades, for joint arthroplasties, spinal and maxillofacial reconstructions, and dental prostheses. However, despite the quite satisfactory survival rates failures still exist. New Ti-alloys and surface treatments have been developed, in an attempt to overcome those failures. This review provides information about new Ti-alloys that provide better mechanical properties to the implants, such as superelasticity, mechanical strength, and corrosion resistance. Furthermore, in vitro and in vivo studies, which investigate the biocompatibility and cytotoxicity of these new biomaterials, are introduced. In addition, data regarding the bioactivity of new surface treatments and surface topographies on Ti-implants is provided. The aim of this paper is to discuss the current trends, advantages, and disadvantages of new titanium-based biomaterials, fabricated to enhance the quality of life of many patients around the world.

Genotoxic and cytotoxic effects of different types of dental cement on normal cultured human lymphocytes

In this study we have investigated the genotoxic and cytotoxic effects of eluates derived from different types of commercially available dental cements, including glass ionomer cements (GICs) (Ketac Cem/3M ESPE and GC Fuji I/GC Corp), resin-modified glass ionomer cements (RM-GICs) (RelyX Luting/3M ESPE and Vitrebond/3M ESPE) and dual-cure resin cements (RCs) (Variolink II/ Ivoclar-Vivadent and Panavia F 2.0/Kuraray) on normal cultured human lymphocytes. Lymphocyte primary cultures obtained from blood samples of three healthy donors were exposed to serial dilutions of eluates derived from specimens of each material tested. Metaphases were induced with phytohaemagglutinin, collected after 72h treatment by use of colchicine and stained according to the fluorescence plus giemsa (FPG) procedure. Preparations were scored for sister chromatid exchange (SCE) and chromosomal aberrations (CAs), while the proliferation rate index (PRI) was also calculated. Our results show that eluates derived from the RM-GICs and RCs caused severe genotoxic effects by significantly increasing the frequencies of SCEs and CAs in cultures of peripheral blood lymphocytes and by decreasing the relevant PRI values in a dose-dependent manner, whereas the two GICs caused only minor cytogenetic effects. Eluates of the two RM-GICs (Vitrebond and RelyX) were also very cytotoxic, as the first serial dilutions of both materials caused a complete mitotic arrest in lymphocyte cultures. Overall, the degree of genotoxicity and cytotoxicity caused by dental cements decreased as follows: Viterbond>Rely X>Panavia F 2.0>Variolink II>Ketac Cem=GC Fuji I. These results indicate that different types of dental cement differ extensively in their genotoxic and cytotoxic potential and their ability to affect chromosomal integrity, cell-cycle progression, DNA replication and repair. Although these results cannot be directly extrapolated to the clinical situation, the potential occurrence of adverse effects caused by the RM-GICs and RCs tested in this study should be considered when making a clinical decision about dental cements.

Comparative characterization of STRO-1neg/CD146pos and STRO-1pos/CD146pos apical papilla stem cells enriched with flow cytometry

OBJECTIVE Stem Cells residing in the Apical Papilla (SCAP) of human permanent teeth represent a promising cell source for dental tissue regeneration. Therefore, the functional and molecular properties of specific subpopulations existing within heterogeneous cultures should be further investigated to give insight whether their selection could be beneficial for targeted therapeutic applications. DESIGN In this study we extensively characterized SCAP cultures established from 10 healthy subjects, as well as their STRO-1(pos/)CD146(pos) and STRO-1(neg/)CD146(pos) subpopulations isolated with fluorescence-activated cell sorting. SCAP were analyzed for embryonic (Nanog, Oct3/4, SSEA-3, TRA-1-60), mesenchymal (STRO-1, CD146/MUC18, CD105/endoglin, CD24, CD90/Thy-1, CD81-TAPA, CD34, CD49f/a6-integrin), neural (CD271/NGFR, nestin) and hematopoietic (CD117/c-kit, CD45) stem cell (SC) markers using flow cytometry. Multipotentiality was evaluated with culture specific staining (Alizarin-Red-S, Oil- Red-O) and RT-PCR analysis for osteo/odontogenic (DSPP, BSP, ALP, osteocalcin, osteonectin, BMP-2, Runx2), adipogenic (lipoprotein-lipase-LPL) and neurogenic (Neurofilament/NFL-L, nestin, β-tubulin-III, NCAM) markers. RESULTS Our results showed that the STRO-1(pos)/CD146(pos) subpopulation demonstrated higher CFU efficiency and much higher expression of several embryonic and mesenchymal SC markers compared to the non-sorted SCAP. They also showed enhanced odontogenic differentiation potential, as evidenced by higher mineralization capacity and expression of osteo/odontogenic markers. By contrast, absence of STRO-1 in the STRO-1(neg)/CD146(pos) subpopulation yielded the opposite results and was associated with significant downgrading of the above-mentioned properties. CONCLUSIONS These results suggest that STRO-1(pos)/CD146(pos) SCAP cells represent a very promising adult MSCs source with enhanced multipotent SC properties that could be easily isolated with simple flow cytometric methods to be used for tissue engineering applications.

Effects of resinous monomers on the odontogenic differentiation and mineralization potential of highly proliferative and clonogenic cultured apical papilla stem cells

OBJECTIVE The aim of this study was to investigate the effects of resinous monomers on the odontogenic differentiation and mineralization potential of apical papilla stem cells (SCAP). METHODS Cultures were established from developing third molars of healthy donors aged 14-18 years-old and were extensively characterized for proliferation rate, colony forming unit efficiency and expression of stem cell markers (STRO-1, CD146, CD34, CD45, CD105, CD117-c-Kit, CD24, CD90, Nanog, Oct3/4), in order to select those with enhanced stem cell and odontogenic differentiation properties. SCAP enriched cultures were then induced for odontogenic differentiation in the continuous presence of low concentrations (0.05-0.5 mM) of the monomers 2-hydroxy-ethyl-methacrylate-HEMA and triethylene-glycol-dimethacrylate-TEGDMA for 3 weeks (long-term exposure). Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were evaluated. RESULTS The results showed that both types of monomer-exposure significantly delayed the odontogenic differentiation and mineralization processes of SCAP cells. A down-regulation followed by recovery in the expression of differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP was recorded. This was accompanied by reduction of the mineralized matrix produced by monomer-treated-compared to non-treated contol cultures. Furthermore, a concentration-dependence was observed for both monomers during long-term exposure, whereas the effects of HEMA were evident at much lower concentrations compared to TEGDMA. SIGNIFICANCE These findings suggest that resinous monomers can delay the odontogenic differentiation of SCAP cells, potentially disturbing the physiological repair and/or developmental processes of human permanent teeth.

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

Genotoxic, cytostatic, antineoplastic and apoptotic effects of newly synthesized antitumour steroidal esters

In this study, we have investigated the genotoxic, cytostatic, antineoplastic and apoptotic effects of three newly synthesized modified steroidal esters, having as alkylating agent p-N,N-bis(2-chloroethyl) aminophenyl butyrate (CHL) or p-N,N-bis(2-chloroethyl) aminophenyl acetate (PHE) esterified with the steroidal nucleus modified in the B- and D-ring. The genotoxic and cytotoxic effects of the compounds were investigated both in vitro, in lymphocyte cultures obtained from blood samples of healthy donors and in vivo, in ascites cells of P388 leukemia obtained from the peritoneal cavity of DBA/2 mice. Preparations were scored for sister-chromatid exchange (SCE) and proliferation-rate indices (PRI). The newly synthesized compounds were also studied for antineoplastic activity against lymphocytic P388 and lymphoid L1210 leukemias in mice, by calculating the mean of the median survival of the drug-treated animals (T) versus the untreated control (C) (T/C%). The activity of caspase-2 and caspase-3, indicators of apoptosis, was assessed biochemically in primary cultures of human lymphocytes. Our results show that the newly synthesized compounds caused severe genotoxic effects by significantly increasing the frequency of SCE and decreasing the PRI values in cultures of peripheral lymphocytes in vitro and in ascites cells of lymphocytic P388 leukemia in vivo. A significant correlation was also observed in both the in vitro and in vivo experiments: the higher the SCE frequency the lower the PRI value (r=-0.65, P<0.001 and r=-0.99, P<0.01, respectively). The measured antileukemic potency was statistically increased by all test compounds in both types of tumours, while the activity of caspase-2 and caspase-3 showed a statistically significant increase after two periods of exposure. The genotoxic (increase of SCE), cytostatic/cytotoxic (decrease of PRI) and antileukemic effects (increase of T/C%) in combination with the induction of apoptosis (activation of caspase-2 and caspase-3) caused by the newly synthesized compounds, lead us to propose them as agents with potentially antineoplastic properties.

Minor salivary glands of the lips: a novel, easily accessible source of potential stem/progenitor cells

ObjectivesCells with stem/progenitor properties have been detected in major salivary glands, but no data are available on their presence within minor salivary glands (MSGs). This study aimed to isolate and characterize potential stem/progenitor cells from human MSGs.Materials and methodsMSGs of the lower lip were surgically obtained during biopsy for Sjogren’s syndrome investigation that finally proved to be histologically normal. The established MSG cultures were assessed for morphology, proliferation, colony-forming-unit efficiency, multipotentiality, and immunophenotypic characteristics.ResultsA mixed population of fibroblast-like and a few flat-shaped epithelial-like cells was obtained. These cells were capable for osteogenic, adipogenic, and neurogenic differentiation. Evidence for strong stem cell potency was observed by the detection of early stem cell markers, like Nanog, Oct-3/4, and SSEA-3. These cells also expressed characteristic mesenchymal stem cell markers, including CD90-Thy1, CD105, CD49f, CD81, nestin, CD146, and Stro-1, but were negative for CD117/C-KIT, CD45, and CD271/NFG. In addition, positivity for keratins 7/8 in part of the population was indicative of an epithelial phenotype, whereas these cells were negative for aquaporin-1 expressed in acinar/myoepithelial cells during development.ConclusionsBased on these data, a cell population with stem/progenitor characteristics was primarily isolated from labial MSGs. The morphologic and immunophenotypic features indicated that this population is mixed with mesenchymal (mainly) and epithelial characteristics.Clinical relevanceDue to their large number and superficial distribution in labial mucosa, MSGs may be proposed as a potential easily accessible source of adult stem/progenitor cells for regenerative therapies of glandular organs with parenchymal pathology.