Expression of procollagen a1 type I and tenascin proteins induced by HEMA in human pulp fibroblasts Michela Zago a , Gabriella Teti a , Giovanni Mazzotti a , Alessandra Ruggeri a , Lorenzo Breschi b , Susi Pelotti c , Michela Ortolani a , Mirella Falconi a, * a Department of SAU&FAL, University of Bologna, Via Irnerio, 48, 40126, Bologna 40126, Italy b Division of Dental Sciences and Biomaterials, Department of Biomedicine, University of Trieste, Trieste 34100, Italy c Department of Medicine and Public Health, Section of Legal Medicine, University of Bologna, Bologna 40126, Italy article info Article history: Received 25 October 2007 Accepted 11 March 2008 Available online 27 March 2008 Keywords: Biocompatibility HEMA monomer Human pulp fibroblasts Tenascin Procollagen a1 type I abstract In the dental pulp extracellular matrix, the main macromolecules are collagenous proteins, non-collage- nous proteins and proteoglycans. Regulated synthesis of the interstitial collagens, in particular, type I col- lagen, is important during development and wound healing but also in a number of pathological conditions. Tenascin is also a matrix protein highly expressed during development while it decreases in mature organs. Under pathological conditions such as infections and inflammation, during tumorigen- esis and mechanical stress applied to cells in culture or tissue in vivo, the expression of tenascin is increased. In this study, HEMA, widely used in dentistry, ophthalmology and drug delivery, has been used to study its influence on the expression of procollagen a1 type I and tenascin proteins in the primary cultures of human pulp fibroblasts. Different concentrations of the resin monomer and different times of exposition were tested. The influence of HEMA on the cell viability was evaluated by means of an MTT assay while immunofluorescence and western blotting analysis were performed to detect possible interference with the presence and the synthesis of these proteins. We observed a strong reduction in cell viability in specimens treated for 96 h and 168 h, especially at con- centrations of 1 and 3 mmol/L HEMA. Both immunofluorescence and western blotting analysis demon- strated a reduction of procollagen a1 type I protein and an overexpression of tenascin protein. Our results showed that long-term exposure and low concentrations of HEMA influence normal cell activ- ity, such as the synthesis of some of the dental pulp extracellular matrix proteins. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Dental pulp is a connective tissue having an unusual organiza- tion and location. It is composed of cells (fibroblasts, odontoblasts and undifferentiated mesenchymal cells) in contact with a com- plex chain of macromolecules secreted in the extracellular matrix (ECM) (Linde, 1985). The main ECM macromolecules are collage- nous proteins especially type I and III collagen, non-collagenous proteins such as fibronectin, tenascin, osteonectin and osteocalcin, and glycosaminoglycans including hyaluronic acid, chondroitin sulfate, heparin sulfate, and phospholipids (1–4). Collagen makes up nearly 34% of the total ECM proteins, and type I and III collagens are the most predominant types. Type I collagen, most commonly found in dense connective tissue, is necessary for tissue architec- ture stabilization (Shuttleworth et al., 1980). Regulated synthesis of interstitial collagens, in particular type I collagen, is important during development and wound healing but also in a number of pathological conditions. Studies on these diseases, as well as vari- ous in vitro models, have shown that collagen synthesis and depo- sition are influenced by cytokines, growth factors and mechanical tension. Inflammatory mediators are important regulatory factors for collagen synthesis in adults (Gressner and Bachem, 1994; Pelto- nen et al., 1991). Tenascin is a matrix protein and the regulation of its expression is poorly understood. It is highly expressed during development while it is quite reduced in developed organs; it reappears under pathological conditions caused by infections and inflammations, during tumorigenesis (Chiquet-Ehrismann and Chiquet, 2003) and mechanical stress applied either to cells in culture or to tissues in vivo (Chiquet-Ehrismann et al., 1994; Fluck et al., 2000). Dental pulp is one of the first tissues of the oral cavity to be in- volved in damage induced by dental restorative processes in which resin-based materials are utilized. During these restorative processes, resin-based materials are applied to the dentin, and the monomers released from the 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.03.008 * Corresponding author. Tel.: +39 051 2091511; fax: +39 051 251735. E-mail address: mirella.falconi@unibo.it (M. Falconi). Toxicology in Vitro 22 (2008) 1153–1159 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit