Superior Survival and Durability of Neurons and Astrocytes on 3-Dimensional Aragonite Biomatrices HAGIT PERETZ, M.S., 1,2 ADOLFO E. TALPALAR, M.D., Ph.D., 1,2 RAZI VAGO, Ph.D., 3 and DANNY BARANES, Ph.D. 1,2 ABSTRACT Current needs of central nervous system therapy urge for the identification of scaffolds supporting the generation and long-term maintenance of healthy and functional neuronal tissue. We compared for the first time the viability of hippocampal neurons and astrocytes grown on conventional 2-dimensional (2D) conditions with that of cells grown on an aragonite bioactive 3-dimensional (3D) scaffold prepared from coralline exoskeleton. Cultures in 3D showed significantly lower mortality rate and higher neurons/ astrocytes ratio than 2D cultures. Moreover, whereas cell survival in 2D was arrested in the absence of the supporting substrates poly-D-lysine and laminin, these substrates had negligible effect on the 3D cultures. Furthermore, aragonite matrices supported cell survival and growth under conditions of cal- cium and nutrients deprivation, whereas in 2D such treatments led to death of all neurons and of almost all astrocytes. To show that the aragonite matrices are permissive for neural cells also in vivo, aragonite matrices having no substrate coating grafted into postnatal rat cortex were invaded by neurons growing on the surface and in multilayer structures resembling those seen in the 3D culture in vitro. Hence, culture of neurons and astrocytes on 3D aragonite coralline matrices is a novel mean for production of stable neuronal tissue, with significant implication to the field of neuronal tissue restoration. INTRODUCTION T HREE-DIMENSIONAL (3D) MATRICES are significant for pe- ripheral and central nervous system regeneration as potential bridge materials, and as templates for tissue de- velopment in vitro. 1–5 In the face of stress conditions at the injury or disease site, like loss of growth factors and break- down of connective tissue and extracellular matrix, 6–8 it is essential that the scaffold be capable of supporting cell ad- hesion and survival even under extreme conditions. It has been shown that culture of hematopoietic cells on tantalum- coated porous biomaterial in 3D promoted long-lasting survival of these cells even in the absence of cytokines, conditions that are devastating for these cells when grown 2 dimensionally 2D. 9,10 Such findings indicate that 3D ma- trices may offer advantages in the long-term preservation of neurons and glia ex vivo. One potential type of scaffold that can meet the need for long-term cell survival and durability is the exoskeleton of some coral species. This group of natural materials, in the form of crystalline aragonite (calcium carbonate), has been shown to be biocompatible and biodegradable and has thus been used successfully in grafting applications. 11–13 Because coralline skeletal materials exhibit a wide range of pore sizes and have a complex surface area because of their needlelike aragonite crystalline topology, they can serve as suitable scaffolds to support the anchorage and growth of a variety of cell types. 14 We have recently demonstrated that aragonite 1 Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel. 2 The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer Sheva, Israel. 3 Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer Sheva, Israel. TISSUE ENGINEERING Volume 13, Number 3, 2007 # Mary Ann Liebert, Inc. DOI: 10.1089/ten.2005.0522 461