Matricellular Protein Expression and Cell Ultrastructure as Parameters to Test In Vitro Cytotoxicity of a Biomimetic Scaffold

Following scaffold implantation, cell sufferance, in-vivo encapsulation, foreign body reaction and inflammatory 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 flasks and on hydrogel scaffold. Based on our results, we propose matricellular protein expression as parameter for testing in vitro biocompatibility of implant materials.