in vitro degradation and cytocompatibility evaluation of novel soy and sodium caseinate-based...

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JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE 14 (2003) 1055±1066 In vitro degradation and cytocompatibility evaluation of novel soy and sodium caseinate- based membrane biomaterials G. A. SILVA 1,2 * , C. M. VAZ 1 , O. P. COUTINHO 2,3 , A. M. CUNHA 1 , R. L. REIS 1,2 1 Department of Polymer Engineering, University of Minho, Campus de Azure Âm, 4800-058 Guimara Äes, Portugal E-mail: [email protected] 2 3B's Research Group, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal 3 Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal Soy- and casein-based membranes are newly proposed materials disclosing a combination of properties that might allow for their use in a range of biomedical applications. Two of the most promising applications are drug delivery carrier systems and wound dressing membranes. As for all newly proposed biomaterials, a cytotoxic scanning must be performed as a preliminary step in the process of the determination of the compatibility with biological systems (biocompatibility). In this study, the cytotoxicity of both soy- and casein- based protein biomaterials has been evaluated and correlated with the materials degradation behavior. It was possible to show, through morphological and biochemical tests that these natural origin materials do not exert any cytotoxic effect over cells, and in some cases can in fact enhance cell proliferation. The different treatments to which the membranes were subjected during their processing (that include crosslinking with glyoxal and tannic acid, and physical modi®cation by thermal treatment) seemed to have a clear effect both on the materials mechanical properties and on their in vitro biological behavior. # 2003 Kluwer Academic Publishers 1. Introduction Natural origin biomaterials are currently under study for several biomedical applications, due to their advanta- geous properties, namely their degradability, availability and renewable origin. Furthermore, they are easily to be processed (both by solvent- and melt-based routes), which results in low cost products. Naturally derived materials offer many mechanical, chemical and bio- logical advantages over synthetic materials, making them suitable for tissue engineering applications [1]. For instance, starch-based materials have already proven to be suitable for several biomedical applications, that range from drug delivery systems, to hydrogels, bone cements and tissue engineering scaffolds in several studies, both in vitro and in vivo [2±7]. Their biocompatibility has already been proved, as docu- mented elsewhere [4, 8, 9]. In this work, soy protein, the major component of the soybean [10] and casein, the major protein present in milk and other biological products, have been studied. Both materials are known proteins that have previously shown [10±12] to have good mechanical, dynamical and thermal properties, which make them suitable for several biomedical applications. Proposed applications include drug delivery carrier systems and wound dressing membranes. The amino acids that constitute soy protein are aspartic and glutamic acid, together with non-polar amino acids such as glycine, alanine, valine and leucine, and basic aminoacids: lysine, arginine and also cysteine, which is present at 5 1% [10]. Milk has a protein content of about 33 g/l and the two major protein fractions are caseins (about 80%) and whey proteins (about 20%) [13, 14]. Caseins are predominantly phosphoproteins that precipitate at pH 4.6 (20 C) and, which are characterized by an open, random coil structure [13, 14]. By treating acid-precipitated caseins with alkali solutions caseinates are produced [13, 14]. Caseins and caseinates form transparent and ¯exible ®lms from aqueous solutions without treatment due to their random coil nature and numerous hydrogen bonds [13, 14]. It is not surprising that caseins and soy have shown to be useful proteins for the microencapsulation of insecticides [15], food ingredients [16] and pharmaceutics [12, 17]. All materials that are aimed to be used in biomedical applications must be evaluated for their biocompatibility, both in vitro and in vivo. The ISO/EN 10993-5 guidelines [18] rule the types of tests to be performed for the evaluation of the proposed biomaterials. In the present work, the cytocompatibility of soy- and casein-based membranes was determined in accordance with these *Author to whom all correspondence should be addressed. 0957±4530 # 2003 Kluwer Academic Publishers 1055

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