Gellan gum: A new biomaterial for cartilage tissue engineering applications J. T. Oliveira,1,2 L. Martins,3 R. Picciochi,1,2 P. B. Malafaya,1,2 R. A. Sousa,1,2 N. M. Neves,1,2 J. F. Mano,1,2 R. L. Reis1,2 13B’s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimara˜es, Portugal 2IBB - Institute for Biotechnology and Bioengineering, PT Associated Laboratory, Guimara˜es, Portugal 3Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, 4710-057 Braga, Portugal Received 26 September 2008; revised 5 May 2009; accepted 6 May 2009 Published online 5 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32574 Abstract: Gellan gum is a polysaccharide manufactured 368C, exhibiting a gelation time of 11 s. Evaluation of by microbial fermentation of the Sphingomonas paucimobilis the gellan gum hydrogels biological performance was microorganism, being commonly used in the food and performed using a standard MTS cytotoxicity test, pharmaceutical industry. It can be dissolved in water, and which showed that the leachables released are not when heated and mixed with mono or divalent cations, deleterious to the cells and hence were noncytotoxic. forms a gel upon lowering the temperature under mild Gellan gum hydrogels were afterwards used to encapsu- conditions. In this work, gellan gum hydrogels were late human nasal chondrocytes (1 3 106 cells/mL) and analyzed as cells supports in the context of cartilage regen- culture them for total periods of 2 weeks. Cells viability eration. Gellan gum hydrogel discs were characterized was confirmed using confocal calcein AM staining. in terms of mechanical and structural properties. Histological observations revealed normal chondrocytes Transmission electron microscopy revealed a quite homo- morphology and the obtained data supports the claim geneous chain arrangement within the hydrogels matrix, that this new biomaterial has the potential to serve as a and dynamic mechanical analysis allowed to characterize cell support in the field of cartilage regeneration. the hydrogels discs viscoelastic properties upon compres- Ó 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: sion solicitation, being the compressive storage and loss 852–863, 2010 modulus of 40 kPa and 3 kPa, respectively, at a frequency of 1 Hz. Rheological measurements determined Key words: hydrogel; natural origin; polysaccharide; the sol-gel transition started to occur at approximately cartilage; tissue engineering INTRODUCTION quality of life. Cartilage is one of the most studied tissues in this field giving the importance it has on Tissue engineering has been proposed as a new mobility and locomotion. Because of its limited method to address problems such as organ failure capacity for self repair, cartilage becomes an enor- and tissue regeneration, being widely studied nowa- mous constraint to normal everyday life once degen- days as a tool to tackle problems in a diverse range erated or traumatized. Structures that can provide of tissues.1–4 Such conditions pose serious health support for specific cells to develop and generate a problems, being responsible for a decrease in people functional cartilaginous tissue are an important sub- ject of study. Different types of natural and synthetic Correspondence to: J. T. Oliveira; e-mail: joao.oliveira@ biomaterials have been processed using different dep.uminho.pt techniques for this purpose. We are proposing in Contract grant sponsor: The Portuguese Foundation for Science and Technology (FCT); contract grant number: this work a new biomaterial-Gellan gum to be used SFRH/BD17135/2004 in the engineering of cartilaginous tissues, even Contract grant sponsor: The European NoE EXPERTIS- though its application may not be restricted to this SUES; contract grant number: NMP3-CT-2004-500283 tissue only, as it will be shown by the different 3D Contract grant sponsor: The European Project HIPPO- CRATES; contract grant number: STRP 505758-1 structures that can be obtained. Recent work per- formed by Smith et al. has also suggested the use of 5 Ó 2009 Wiley Periodicals, Inc. this biomaterial for tissue engineering applications. GELLAN GUM FOR CARTILAGE TISSUE ENGINEERING APPLICATIONS 853 Gellan gum is a linear anionic polysaccharide bonding between divalent cations and two carboxy- composed of tetrasaccharide (1,3-b-D-glucose, 1,4-b-D- late groups belonging to glucuronic acid molecules glucuronic acid, 1,4-b-D-glucose, 1,4-a-L-rhamnose) in the Gellan chains, in adittion to the screening repeating units, containing one carboxyl side group, effect.19 It was also suggested that different types of and was initially described by Moorhouse et al.6,7 mono or divalent cations also influenced the visco- þ This material has a broad use in the food industry elastic behavior of Gellan gum solutions. K was þ þ and biomedical fields, mostly due to its processing more remarkable than Na , and Ca2 more than þ into transparent gels that are resistant to heat and Mg2 .11 Gellan gum structures have excellent heat acid stress. Two gellan gum forms exist, acetylated resistance properties because the formed junctions and deacetylated, being the latter the most common upon gelation can only be unzipped on heating at and commercially available form. Both form thermor- 1208C.11 In the initial state, a junction zone in Gellan eversible gels, varying in their mechanical properties gum is estimated to be four double helices wide and from soft and elastic for the acetylated form to hard five repeat units long, its length being increased to and brittle for the fully deacetylated polysaccha- seven repeat units upon annealing.10 In the solid ride.8,9 Gellan gum can form gels in the following state, the double helix structure adopted by Gellan way: at high temperatures, Gellan gum is in the coil gum has a similar arrangement to the double helix form; upon temperature decrease, a thermally- structure of iota carrageenan.20 Previous studies reversible coil to double-helix transition occurs, which indicate that solutions of deacetylated gellan gum is a prerequisite for gel formation. Afterwards, a behave as a pseudoplastic liquids, as evidenced by structure composed of anti-parallel double helices self creep testing, and have little thixotropy.13 Gellan assembled to form oriented bundles, called junction gum advantageous use in the context of biomedical zones, is formed. Untwined regions of polysaccharide applications includes its lack of toxicity, processing chains, in the form of extended helical chains, link the under mild conditions, the ability to used as an junction zones, leading to the formation of a three injectable system in a minimally invasive manner, dimensional network, that creates the gel.10 These and also the structural similarity it presents with structural changes occurring to gellan gum molecules native cartilage glycosaminoglycans by the presence have been shown by different techniques. During the of glucuronic acid residues in their repeating cooling process, for example, rheological and differ- unit.21,22 The presence of this carbohydrate residue, ential scanning calorimetry (DSC) studies revealed a which contains carboxylic groups, may confer added first step increase of loss modulus that corresponds to functions to this material. Some intellectual property the coil-helix transition, and a second step increase of associated with the application of this material in the loss modulus due to sol-gel transition.11 The gelation medical field has already been disclosed, as its use of gellan gum solutions is strongly influenced by the for ophthalmologic purposes.23,24 chemical nature and quantity of cations present in the This work tested for the first time gellan gum as a solution. The presence of cations is critical when a new biomaterial to be used in cartilage regeneration structurally stable gel is to be prepared.10,12,13 In fact, approaches. As shown here, gellan gum hydrogels at low Gellan gum concentrations, the helix formation are quite versatile in terms of processing and its and its partial aggregation may form an ordered materials properties reveal good prospects for their structure, but this does not lead to gel formation use as a cell encapsulating agents. Biological evalua- because the number of helical aggregates does not tion of their cytotoxicity and in vitro culturing of give rise to a continuous network in the whole human nasal chondrocytes generated interesting volume.11 The main barrier are the carboxyl side results indicating that this new biomaterial may play groups that repulse each other by electrostatic interac- a potential role in cartilage regeneration approaches. tion, therefore hindering the tight binding of helices and their cohesive aggregation.9,14–16 The introduc- tion of cations shields the electrostatic repulsion and MATERIALS AND METHODS thereby allows the tight binding and aggregation of helices.11,17,18 The gelation properties of Gellan gum are also Versatility of Gellan gum: Processing into different structures (discs, membranes, fibers, influenced by the nature of the cations used, in particles, scaffolds) which divalent cations promote the gelation much more strongly than monovalent cations.11,12 In mono- Note: Unless otherwise stated the reagents were valent cations, the gelation is mainly a result of the purchased from Sigma-Aldrich. screening of the electrostatic repulsion between the Gellan gum (G1910, Sigma, St. Louis, MO) was ionized carboxylate groups on the Gellan gum processed in different ways giving rise to various struc- chains. In the case of divalent cations, the gelation tures, therefore evidencing the versatility of this natural bio- and aggregation of Gellan occurs via a chemical material. The processing involved temperature-dependent Journal of Biomedical Materials Research Part A 854 OLIVEIRA ET AL. and pH-dependent reactions. Regarding gellan gum discs tetroxide for 2 h at room temperature and then washed and membranes production, the following methodology 3 times in PBS.