Available online at www.sciencedirect.com Journal of Biotechnology 132 (2007) 218–226 In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes T.C. Santos a,b , A.P. Marques a,b , S.S. Silva a,b , J.M. Oliveira a,b , J.F. Mano a,b , A.G. Castro c , R.L. Reis a,b,∗ a 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, Campus de Gualtar, 4710-057 Braga, Portugal b IBB – Institute for Biotechnology and Bioengineering, Braga, Portugal c Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal Received 2 January 2007; received in revised form 22 June 2007; accepted 6 July 2007 Abstract Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan- based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O 2 - and HO - and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O 2 - or HO - . Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications. © 2007 Published by Elsevier B.V. Keywords: Chitosan; Soy; Lysozyme; Reactive oxygen species; Neutrophils; Wound healing 1. Introduction In the past few years a huge effort has been made to cre- ate the ideal wound dressing and skin substitutes to respond to the increasing needs of mankind. Several research groups suggested that chitosan is a promising material for regenera- ∗ Corresponding author at: 3B’s Research Group – Biomaterials, Biodegrad- ables and Biomimetics, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Tel.: +351 253604781; fax: +351 253604498. E-mail address: ruigreis@dep.uminho.pt (R.L. Reis). tive medicine (Shi et al., 2006; Marreco et al., 2004; Azad et al., 2004; Khor and Lim, 2003). Chitosan-based membranes were shown to promote the proliferation of human skin fibrob- lasts and keratinocytes in vitro (Azad et al., 2004) and the application of a chitosan-based hydrogel in mice wounds had proved to accelerate wounding (Ishihara et al., 2001). Further- more it was demonstrated that wounds in human skin heal better if covered with chitosan-based membranes (Howling et al., 2001; Wedmore et al., 2006), beyond its proven antimicro- bial properties (Burkatovskaya et al., 2006; Singla and Chawla, 2001). 0168-1656/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.jbiotec.2007.07.497