Please cite this article in press as: M. Temtem, et al., Supercritical CO 2 generating chitosan devices with controlled morphology. Potential application for drug delivery and mesenchymal stem cell culture, J. Supercrit. Fluids (2008), doi:10.1016/j.supflu.2008.10.020 ARTICLE IN PRESS G Model SUPFLU-1684; No. of Pages 9 J. of Supercritical Fluids xxx (2008) xxx–xxx Contents lists available at ScienceDirect The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu Supercritical CO 2 generating chitosan devices with controlled morphology. Potential application for drug delivery and mesenchymal stem cell culture Márcio Temtem a , Lígia M.C. Silva a , Pedro Z. Andrade b , Francisco dos Santos b , Cláudia Lobato da Silva b , Joaquim M.S. Cabral b , Manuel M. Abecasis c , Ana Aguiar-Ricardo a,∗ a REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal b IBB-Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal c Bone Marrow Transplantation Unit, Instituto Português de Oncologia, R Prof Lima Basto 1093, Lisboa Codex, Portugal article info Article history: Received 22 August 2008 Received in revised form 19 October 2008 Accepted 20 October 2008 Keywords: Supercritical carbon dioxide Membranes Chitosan Drug delivery and mesenchymal stem cells abstract In this work, a novel approach involving supercritical carbon dioxide (scCO 2 ) induced phase inversion technique was developed to produce chitosan devices using moderate temperatures and three very envi- ronmentally acceptable solvents (water, ethanol and CO 2 ). The morphology and three-dimensional (3D) structure were controlled by altering the co-solvent (ethanol) composition in the carbon dioxide non- solvent stream during the demixing induced process. Microarchitectural analysis by scanning electron microscopy identified the production of particulate agglomerates when 10% of ethanol in the scCO 2 stream was used and the ability to make porous membranes with different morphologies and mechanical prop- erties depending on the programmed gradient mode and the entrainer percentage (2.5–5%) added to the scCO 2 stream. These structures were characterized in terms of pure water flux, porosity, mechanical prop- erties and biodegradability. These chitosan matrices exhibited low solubility at neutral pH conditions, with no further modifications. We also demonstrated that the current method allows for a single-step prepara- tion of an implantable antibiotic release system by co-dissolving gentamicin with chitosan and the solvent. Finally, the cytotoxicity, as well as the ability of these structures to support the adhesion and prolifera- tion of human mesenchymal stem cells (MSC) in vitro were also addressed. The studies described may provide a starting point for the “green” design and production of chitosan-based materials with potential applications in tissue engineering and regenerative medicine, as well as drug delivery. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Biocompatible and biodegradable polymers have various impor- tant applications in the biomedical field particularly in the development of supports for tissue repair and regeneration [1]. These structures have a high level of porosity, with a good intercon- nectivity among the pore network system together with significant mechanical strength and flexibility. Membranes are the most widely studied scaffolds for guided bone regeneration. They are useful for repair in sites where limited mechanical loading exists, for example, in some cranial or maxillofacial areas, in dental appli- cations [2] and as 3D matrices in perfusion bioreactors [3]. Another important area of application of such materials is in drug delivery. In biomedical applications, residual solvents are undesirable since they may contaminate the device and cause toxicity problems. In ∗ Corresponding author. Fax: +351 212 948 385. E-mail address: aar@dq.fct.unl.pt (A. Aguiar-Ricardo). order to avoid those negative effects, alternative approaches are being developed, namely supercritical fluid processing, of which the most commonly used is carbon dioxide (CO 2 ). Besides the environmental advantages and the additional parameters that can be used to control the morphology (pressure, temperature, and depressurization rate), scCO 2 presents liquid-like densities and gas- like viscosities and diffusivities, ideal for penetrating into porous structures. In addition, being a gas at normal conditions, CO 2 can easily be removed from the pores without leaving any solvent residues. Importantly, scCO 2 technology potentially allows the pro- duction of sterile, ready-to-use devices, due to the high pressure features of this technique. In fact, reports in the literature suggest that scCO 2 can be potentially used for sterilization of biomedical devices, being effective against bacteria [4], viruses [5] and spores [6], although additives are required in order to achieve terminal sterilization, namely hydrogen peroxide [6] or peracetic acid [7].A complete review concerning the potentialities of supercritical flu- ids in the processing of polymer systems for drug delivery, as well as tissue engineering/regenerative medicine can be found in literature [8]. During the last decade several techniques were developed for 0896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2008.10.020