Expression of procollagen type I, MIP-1alpha and SDF-1alpha by human gingival fibroblasts and dental pulp stimulated by Enterococcus faecalis lipoteichoic acid and lipopolysaccharide from Escherichia coli and Porphyromonas gingivalis

Abstract

Dental pulp is a connective tissue structure constituted by many different cell types. Among them, fibroblasts are the most abundant. When challenged by different aggressive agents, these cells are able to release some substances like cytokines and chemokines, which are essential to trigger the inflammatory process. Nevertheless, these cells also produce extracellular matrix compounds, like collagen, essential for tissue repair. Therefore, the aims of this study are to: 1. evaluate type I procollagen expression; 2. evaluate chemokines expression (MIP-1alpha and SDF-1alpha) by human gingival and dental pulp fibroblasts challenged by E. faecalis lipoteichoic acid (LTA) as well as E. coli and P. gingivalis lipopolysaccharide (LPS); 3. compare type I procollagen and chemokines release by fibroblasts when challenged by LTA or both LPS; 4. compare the cellular behavior when originated from dental pulp or from gingiva from the same subject using the same stimuli. The dental pulp from non-erupted third molars and health gingival samples will be collected from the same patient (n=5). Explants will be made and stocked in culture medium for fibroblasts growth. The cells will be used since the fourth passage. In 24-well plates and after cellular confluence, culture medium containing 0.8-80ug/mL of E. faecalis LTA or 1-100ug/mL of E. coli or P. gingivalis LPS will be added. After 1, 6 and 24 hours, the supernatant and the cells will be collected for analysis. Reverse transcription followed by real time polymerase chain reaction will be employed to evaluate mRNA expression for type I procollagen, MIP-1alpha and SDF-1alpha. Protein detection of type I procollagen will be achieved by Western blot analysis, whereas detection of MIP-1alpha and SDF-1alpha will be carried out by ELISA. Statistical analysis will be performed by GraphPad Prism 3.0 software, ANOVA and Bonferroni correction tests. Significance levels of 5% will be adopted. (AU)