Candida bracarensis: Evaluation of virulence factors and its tolerance to Amphotericin B and Fluconazole

Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.This work was supported by the projects PTDC/SAU-MIC/119069/2010, PEst-OE/EQB/LA0023/2013, from Fundação para a Ciência e Tecnologia (FCT), Portugal and ‘‘BioHealth—Biotechnology and Bioengineering approaches to improve health quality’’, Ref. NORTE-07-0124FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors also acknowledge the project ‘‘Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB’’, Ref. FCOMP-01-0124-FEDER027462

HUVECs Synergize Osteo/Odontogenic Differentiation Of PDLSCs In 3D Cell Sheets

Conference Theme: We are the FutureSession 7: O7Oral PresentationObjective: To investigate the expression of osteo/odontogenic differentiation markers and vascular network formation in a 3-D cell sheet with varying cell ratios of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs). Method: Human PDLSCs were isolated and characterized and co-cultured with HUVECs. Both types of cells were seeded on temperature responsive culture dishes with PDLSCs alone, HUVECs alone, and various ratios of these cells (1:1, 2:1, 5:1 and 1:5). The expressions of osteo/odontogenic pathway markers ALP, BSP and RUNX2 were analyzed at 3 and 7 days using RT-PCR. Further, ALP protein quantification was performed at 7 and 14 days using alkaline phosphatase assay. The calcium nodule formation was assessed qualitatively and quantitatively by alizarin red assay. Histological evaluations of cell sheet constructs with combinations (PDLSCs-PDLSCs-PDLSCs/PDLSCs-HUVECs-PDLSCs/Co-Culture-Co-culture-Co-culture) were observed with scanning electron microscope (SEM), H&E and immunofluorescence staining. Cell viability was assessed with the live/dead assay. Statistical analysis was performed using t-test(p<0.05). Result: Significantly higher ALP gene expression was observed at 3days in 1:1 (PDLSCs:HUVECs) (2.52±0.67) and 5:1(4.05±1.07) co-culture groups compared with other groups(p<0.05), being consistent with ALP protein quantification. However, the expression of BSP and RUNX2 genes was higher at 7days compared to 3days. Significant calcium mineralization was detected as quantified by alizarin red assay at 14days in 1:1(1323.55±6.54µm) and 5:1(994.67±4.15µm) co-cultures as compared with mono-culture cell sheets(p<0.05). SEM, H&E and CD31 immuno-staining exemplified the development of a layered cell sheet structure with endothelial cell islands within the constructed PDLSCs-HUVECs-PDLSCs and co-culture groups. HUVECs invaded the layered cell sheet, suggestive of rudimentary vascular network initiation. After 7 days, majority of the cells were viable in all 3 groups. Conclusion: PDLSCs-HUVECs co-culture cell sheet model exhibits significantly high levels of osteo/odontogenic markers with signs of initial vascular formation. This novel 3-D cell sheet-based approach may be potentially beneficial for periodontal regenerative therapy This abstract is based on research that was funded entirely or partially by an outside source: This work is supported by seed funding programme for Basic Research,The University of Hong Kong. 20111115919

Development of Novel 3D Stem Cell Sheet for Periodontal Regeneration

Poster PresentationSession 151: Periodontal Tissue RegenerationObjective: To assess the regenerative capacity of 3D periodontal ligament stem cell (PDLSC) sheet in vivo. Method: Single culture and co-culture cell sheets were prepared by using PDLSCs and Human umbilical vein endothelial cells (HUVECs) in thermo-responsive culture dishes. Scanning electron microscopy (SEM) was used to observe the ultra-structure of single and co-culture groups of cell sheets. Cells were labeled with cell-tracker dyes (PDLSCs-Green, HUVECs-Red) and seeded in different ratios to assess the time dependent modulation of vascular network formation on Matrigel (4 and 8 hours). Tooth root fragments were coated with PDLSC sheets and implanted into the subcutaneous space of SCID mice. After 2 weeks, implants were retrieved and processed for histological evaluation. Result: SEM results showed the ultra-structural arrangement and preserved extracellular matrix (ECM) of layered cell sheet constructs. Furthermore, increased ECM deposition was observed in co-culture groups. PDLSCs and HUVECs seeded on Matrigel showed initiation of vessel like structure formation after 4 hours. Extensive lattice of vessel like structures was formed in 1:2 and 1:5 groups compared to other groups. However, after 8 hours all the groups showed formation of island like cell aggregations. In vivo, H&E sections exemplified the successful integration of implanted PDL cell sheet coated roots with host tissues after 2 weeks. Periodontal ligament-like arrangement was observed around root surface in cell sheet groups compared to control groups. Conclusions: 3D cell sheet model demonstrated a promising regenerative potential in vivo and this approach would be useful in future periodontal bioengineering strategies. This abstract is based on research that was funded entirely or partially by an outside source: Seed Funding for Basic Research 201111159191 The University of Hong Kon