Haruki Nishimura

Effect of serum concentration on Candida biofilm formation on acrylic surfaces

The biofilm formation of the oral fungal pathogen Candida on denture acrylic strips coated with saliva, serum and, saliva–serum pellicle were examined in vitro using Candida albicans (four isolates), Candida glabrata (three isolates) and Candida tropicalis (three isolates). The degree of biofilm activity varied depending upon both the isolate and the pellicle. Significantly increased biofilm activity on the pellicle (particularly serum)‐coated strips was observed with three isolates of C. albicans and another of C. glabrata on protein‐coated acrylics, with increasing concentration of serum in the pellicle. Similar trends were observed with one isolate of C. albicans and C. glabrata, although the effects of pellicles were not significant. In contrast, with all three isolates of C. tropicalis and a single isolate of C. glabrata, although the biofilm activity on the protein‐free control strips was significantly higher than that of saliva‐coated strips, the increase in activity of pellicle‐admixed biofilm depended upon the serum concentration. Candidal biofilm formation on acrylic surfaces is essentially promoted with increasing concentration of serum in the pellicle. This suggests that inflammation in the oral environment would facilitate fungal colonization on denture acrylic.

Effects of modified pellicles on Candida biofilm formation on acrylic surfaces.

The role of saliva or serum proteins, such as mucin, fibronectin (FN) and mannan‐binding protein, on Candida biofilm formation was investigated. Supplementation of saliva with FN had no significant effect on biofilm formation. In contrast, biofilm formation on either mucin‐coated or FN‐coated acrylic surfaces was significantly less than that of the control. These results suggest that salivary mucin or FN alone does not facilitate biofilm formation of Candida. Supplementation of serum with FN increased biofilm formation of C. glabrata compared with the control. Pretreatment of serum with anti‐FN monoclonal antibody significantly reduced biofilm formation, as did pretreatment of serum with anti‐mannan‐binding protein monoclonal antibody or Con‐A. Therefore, Candida biofilm formation on acrylic surfaces appears to be a complex phenomenon involving a multiplicity of proteins operating intraorally.

Relationship between thigmotropism and Candida biofilm formation in vitro

The biofilm formation of the oral fungal pathogen Candida on denture acrylic strips coated with saliva or serum was examined in relation to the ability to induce hyphae by thigmotropic reaction, using C. albicans (4 isolates), C. glabrata (3 isolates) and C. tropicalis (3 isolates). Both the degree of biofilm formation and the amount of hyphae exhibiting thigmotropism varied depending upon both the species and strains of Candida. Although there was no significant correlation between the amount of hyphae induced by thigmotropic reaction of fungal isolates and biofilm formation on uncoated control specimens (r = 0.577; p < 0.05), the ability of hyphae induced by thigmotropic reaction significantly correlated with the amount of both saliva- and serum-admixed biofilms (r = 0.734; p < 0.05 and r = 0.793; p < 0.01, respectively). Taken together our in vitro data suggested that the hyphal induction by thigmotropic reaction is of importance in candidal biofilm formation on saliva- or serum-coated acrylic surfaces.The biofilm formation of the oral fungal pathogen Candida on denture acrylic strips coated with saliva or serum was examined in relation to the ability to induce hyphae by thigmotropic reaction, using C. albicans (4 isolates), C. glabrata (3 isolates) and C. tropicalis (3 isolates). Both the degree of biofilm formation and the amount of hyphae exhibiting thigmotropism varied depending upon both the species and strains of Candida. Although there was no significant correlation between the amount of hyphae induced by thigmotropic reaction of fungal isolates and biofilm formation on uncoated control specimens (r = 0.577; p < 0.05), the ability of hyphae induced by thigmotropic reaction significantly correlated with the amount of both saliva- and serum-admixed biofilms (r = 0.734; p < 0.05 and r = 0.793; p < 0.01, respectively). Taken together our in vitro data suggested that the hyphal induction by thigmotropic reaction is of importance in candidal biofilm formation on saliva- or serum-coated acrylic surfaces.

Bacterial and Candida adhesion to intact and denatured collagen in vitro

Summary.  Several recent reports imply the possibility of cariogenicity and periodontal disease linked to denture plaque containing Candida albicans. Adhesion of oral bacteria and Candida species to the extracellular matrix, such as type I collagen, fibronectin and denatured type I collagen, was examined by using adenosine triphosphate (ATP) analysis. The adhesion of C. albicans to intact and denatured type I collagen was significantly greater than those of oral bacteria and other species of Candida. This result suggests that C. albicans possesses the ability to adhere specifically to extracellular matrix, as compared with other Candida species or oral bacteria.

Quantification of thigmotropism (contact sensing) of Candida albicans and Candida tropicalis

To quantify the thigmotropism, we adapted the our previous method using a chemotaxifilter system in combination with a bioluminescent adenosine triphosphate (ATP) assay based on firefly luciferase-luciferin system and analyzed the relationship between the ability of germ tube formation and thigmotropism of C. albicans and C. tropicalis.Both the ability to form germ tube and the amount of hyphae exhibiting thigmotropism varied depending upon both the species and strains of Candida. C. albicans formed more germ tubes than C. tropicalis. A good correlation was observed between the ability to form a germ tube and the capacity for thigmotropism, and the results gave a level of significance (p<0.05).Further, SEM observation revealed that relatively long hyphae of C. tropicalis with penetrated through the pores of filter membrane. This phenomenon may be of importance in the development of pathogenesis of C. tropicalis as well as C. albicans.

Cell-associated collagenolytic activity by Candida albicans.

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans.

Enhancement of Osteogenesis by Concanavalin A in Human Bone Marrow Mesenchymal Stem Cell Cultures

This study investigates concanavalin A (ConA) as a novel factor that may enhance osteogenesis of mesenchymal stem cells (MSCs) in vitro. Various factors, such as cytokine bone morphogenetic protein-2 (BMP-2), have been studied for their possible promotion of MSC osteogenesis in vivo and in vitro. However, the factor that might be safer, more effective, and less expensive than these has not been determined. We therefore cultured human MSCs in osteogenic medium in the presence or absence of ConA, and used calcium assays to compare the effects of ConA and BMP-2 on MSC calcification. We also used enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (PCR) to evaluate the expression levels of bone-specific markers. ConA and BMP-2 enhanced calcification with comparable effectiveness. The combination of ConA and BMP-2 further enhanced calcification slightly but significantly. ConA also increased osteocalcin and BMP-2 protein levels in MSC culture medium. Furthermore, ConA increased osteocalcin, RUNX2, BMP-2, BMP-4, and BMP-6 mRNA expression levels. However, the gene expression pattern of ConA-stimulated MSCs was different from that of MSCs stimulated by BMP-2. Together, these results suggest that ConA and BMP-2 enhance MSC osteogenesis via different pathways. ConA-induced bone formation in MSC cultures may be useful in regenerative medicine or tissue engineering in clinical studies, as well as in basic research on bone formation.

Bacterial and Candida adhesion to intact and denatured collagen in vitro - Bakterien- und Candida-Adharenz an intaktem und denaturiertem Kollagen in vitro

Several recent reports imply the possibility of cariogenicity and periodontal disease linked to denture plaque containing Candida albicans. Adhesion of oral bacteria and Candida species to the extracellular matrix, such as type I collagen, fibronectin and denatured type I collagen, was examined by using adenosine triphosphate (ATP) analysis. The adhesion of C. albicans to intact and denatured type I collagen was significantly greater than those of oral bacteria and other species of Candida. This result suggests that C. albicans possesses the ability to adhere specifically to extracellular matrix, as compared with other Candida species or oral bacteria.