Volume 5 • Issue 5 • 1000262 J Cytol Histol ISSN: 2157-7099 JCH, an open access journal Research Article Open Access Durante et al., J Cytol Histol 2014, 5:5 http://dx.doi.org/10.4172/2157-7099.1000262 Research Article Open Access Cytology & Histology Matricellular Protein Expression and Cell Ultrastructure as Parameters to Test In Vitro Cytotoxicity of a Biomimetic Scaffold Sandra Durante 1 , Gabriella Teti 1 , Viviana Salvatore 1 , Stefano Focaroli 1 , Monica Mattioli-Belmonte 2 , Concetta Ferretti 2 , Adriana Bigi 3 , Roberto Giardino 4 and Mirella Falconi 1 * 1 Department for Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, via Irnerio 48, Bologna, 40126 Italy 2 Department of Clinical and Molecular Sciences, School of Medicine, Università Politecnica delle Marche, Via Tronto 10/60126, Ancona, Italy 3 Department of Chemistry “G. Ciamician”, University of Bologna, via Selmi 2, 40128 Bologna Italy 4 BITTA Laboratory, Rizzoli Orthopedic Institute, Via di Barbiano 1/10, Bologna, 40136, Italy *Corresponding author: Mirella Falconi, Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, via Irnerio, 48-40126 Bologna (Italy), Tel: +39 051 2091511; Fax: +39 051 251735; E-mail: mirella.falconi@unibo.it Received June 03, 2014; Accepted July 11, 2014; Published July 13, 2014 Citation: Durante S, Teti G, Salvatore V, Focaroli S, Mattioli-Belmonte M, et al. (2014) Matricellular Protein Expression and Cell Ultrastructure as Parameters to Test In Vitro Cytotoxicity of a Biomimetic Scaffold. J Cytol Histol 5: 262. doi:10.4172/2157-7099.1000262 Copyright: © 2014 Durante S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Following scaffold implantation, cell sufferance, in-vivo encapsulation, foreign body reaction and inlammatory response has been reported and the up- regulation of matricellular proteins is often connected with this condition. Cytotoxicity of biomaterials is generally tested according to ISO standard 10993-5 based mainly on viability tests. Additional assays, based on improved cytotoxicity knowledge, are suggested in order to better analyze the biocompatibility of implant materials. The purpose of the study was to evaluate the matricellular protein expression as biomarker for in vitro-testing the biocompatibility of implant materials. Tenascin-C, osteocalcin and osteopontin belong to the matricellular protein family and were chosen as cytotoxicity markers. Mesenchymal stem cells were seeded on collagen/hydroxyapatite scaffold and on carboxymethyl cellulose based hydrogel in order to evaluate gene/protein expression by cell viability test, Real Time PCR and western blot. Electron microscopy was carried out to evaluate the morphological changes induced by cell/scaffold interactions. A low expression of tenascin-c, osteonectin and osteopontin was demonstrated in collagen/hydroxyapatite scaffold compared to the cells cultured on tissue lasks and on hydrogel scaffold. Based on our results, we propose matricellular protein expression as parameter for testing in vitro biocompatibility of implant materials. Keywords: Matricellular proteins; In vitro biocompatibility; Biomaterials; Mesenchymal stem cells; Real Time PCR Introduction Cytotoxicity assays are widely used in in-vitro biocompatibility studies. Currently, the in vitro cytotoxicity of implant materials is being tested according to ISO standard 10993-5 [1], based mainly on viability tests. he lactate dehydrogenase (LDH) leakage, the protein assay, the neutral red and the MTT assays are those most commonly employed in the detection of cytotoxicity or cell viability following exposure to biomaterials [2]. In vitro macrophage responses are widely utilized for inlammation reaction [3]. A diferent sensitivity was observed for each assay with the neutral red and MTT assays being the most sensitive in detecting cytotoxic events as compared to the LDH leakage and the protein assays. Although viability assays can be used to predict the compatibility of medical devices [4], the necessity of more sensitive tests which investigate gene and protein expression in cells grown in contact with biomaterials is strongly recommended in order to predict the behaviour of a implant materials before in vivo testing. he inlammatory response, biological encapsulation and foreign body reaction, and an unusual reaction to injury occurring in vivo ater the implantation of the biomaterials can impair the performance of implanted scafolds [5]. Key molecules which have a role in modulating the in vivo reaction to the implantation of materials have recently been explored and are referred to as matricellular proteins [6]. he matricellular proteins are a group of extracellular matrix (ECM) proteins which are synthesized by a number of diferent cell types including ibroblasts, macrophages and endothelial cells [7]. hey can regulate cell movement, proliferation and diferentiation during morphogenesis and the inlammatory and reparative response following tissue injury and regeneration. he expanding family of matricellular proteins includes, osteonectin (SPARC), osteopontin (OPN) and tenascin-C (TNC) [7]. Abundantly expressed during development, their level is generally low under steady state conditions in adult tissue, but they are readily upregulated in wound healing and tissue remodelling [8-10], conditions which activate an inlammatory response. An aberrant expression of matricellular proteins is oten detected in tumors where they actively contribute to tumor progression and metastasis [11]. he aim of the study was to propose matricellular protein expression, such as TNC, SPARC and OPN, as a potential assay in testing the in vitro biocompatibility of implant materials. he idea was to propose a more in vitro sensitive assay for predicting the behaviour of biomaterials in inducing adverse reactions ater in vivo implantation. Our hypothesis was that elevated expression of matricellular proteins in cells grown on implant materials was the key signal for potential encapsulation, foreign body reaction and the inlammatory response which could strongly impair the eiciency of the material [12]. On the