Development and evaluation of tissue model infection

Purpose. In vitro analyses of virulence, pathogenicity and associated host cell responses are important components in the study of biofilm infections. The Candida-related infection, denture-associated oral candidosis, affects up to 60 % of denture wearers and manifests as inflammation of palatal tissues contacting the denture-fitting surface. Commercially available three-dimensional tissue models can be used to study infection, but their use is limited for many academic research institutions, primarily because of the substantial purchase costs. The aim of this study was to develop and evaluate the use of in vitro tissue models to assess infections by biofilms on acrylic surfaces through tissue damage and Candida albicans virulence gene expression.

Methodology. In vitro models were compared against commercially available tissue equivalents (keratinocyte-only, SkinEthic; full-thickness, MatTek Corporation). An in vitro keratinocyte-only tissue was produced using a cancer-derived cell line, TR146, and a full-thickness model incorporating primary fibroblasts and immortalised normal oral keratinocytes was also generated. The in vitro full-thickness tissues incorporated keratinocytes and fibroblasts, and have potential for future further development and analysis.

Conclusion. Our results confirm the feasibility and suitability of using these alternative in vitro tissue models for such analyses.

Results are provided as microscopy images of sections of tissue constructs, with or without biofilm infection. The sections are stained with haematoxylin and eosin to visualise cellular structure, and identify microorganisms within the tissue layers.

Tissue construct damage is presented graphically as fold change in lactate dehydrogenase (LDH) activity. The collected data includes optical density readings of samples using a spectrophotometer, and represented by raw absorbance values. The fold changes in the quantity of LDH was calculated, and is presented in a table (excel spreadsheet), from which the graphical representation is produced.

The Candida albicans virulence factors, and host cell responses are expressed as fold change in the expression of specific genes. The raw gene expression data is presented as cycle threshold (Ct) values, from which, normalised control values are calculated, and differences between the test conditions are determined (ddCt). These ddCt values are also presented numerically as fold change, and visually represented graphically.

Research results based upon these data are published at http://doi.org/10.1099/jmm.0.000677