Regenerated cellulose scaffolds: Preparation, characterization and toxicological evaluation
Introduction
Cellulose, the most abundant natural polymer on Earth (Klemm, Heublein, Fink, & Bohn, 2005), is a linear homopolysaccharide that represents the largest component of plant biomass. It consists of units of β-d-glucopyranose (β-glucose) linked by glycosidic type β-(1 → 4).
Industrially cellulose is widely used in paper production as emulsifying, dispersing agent, gelling agent, among other functions, with interesting features such as biodegradability, biocompatibility, no toxicity and no allergenicity (Czaja, Krystynowicz, Bielecki, & Brown, 2006). The physicochemical properties of cellulose have attracted great interest in the production of new materials in various areas, such as electronics (LCD screens), energy (fuel cell membranes), communications (diaphragms for microphones and stereo headphones) and medicine (temporary artificial skin for burns and ulcers). In recent years, there is an increase in interest for using cellulose for medical applications, especially in exploring new porous biomaterials, which might be used for tissue engineering (Hench & Polak, 2002).
An ideal biomaterial must provide variety of shapes and sizes, and also be tough enough to be used in locations where there is impact load. Moreover, it must also be biocompatible, absorbable and replaced by new tissue formation, in case of application in tissue engineering (Karageorgiou and Kaplan, 2005, Spector, 2008). Currently, natural polymers are important alternatives in obtaining scaffolds for tissue repair (Liu & Ma, 2004).
Concerning regenerated cellulose, it may be obtained by the well-known viscose rayon process. In short, cellulose pulp is treated with sodium hydroxide and carbon disulfide solutions, leading to cellulose xanthate. Regeneration of cellulose occurs by acid hydrolysis of xanthates groups using sulfuric acid solution. In this step, the xanthate groups are released from the main chain of cellulose and the cellulose is regenerated (Cross et al., 1893a, Cross et al., 1893b).
The applicability of regenerated cellulose in tissue repair is being evaluated. Martson, Viljanto, Hurme, and Saukko (1998) evaluated the biocompatibility of scaffolds based on regenerated cellulose for bone repair in rats. They showed that regenerated cellulose was a compatible and osteoconductive matrix, promoting new bone formation. Cullen et al. (2002) described a new wound treatment using oxidized regenerated cellulose (ORC) and collagen (ORC/collagen). The ORC/collagen composite was able to inactivate potentially harmful factors (proteases, oxygen free radicals and excess metal ions) presented in chronic wound fluid, and therefore promote healing. Additionally, the effectiveness of pharyngeal mucosal healing using ORC was also reported (Liu et al., 2012). Nevertheless, further studies are necessary to establish a comparison among different biomaterials and to evaluate the application of regenerated cellulose scaffolds in clinical practice. Considering that the human body does not express naturally the cellulase enzymes able to degrade cellulose (Hu & Catchmark, 2011a) and in order to obtain in vivo resorption of the cellulose, this study evaluated the in vitro enzymatic degradation for regenerated cellulose scaffolds using two different enzymes, Trichomona reesei cellulase and lysozyme.
Once a material undergoes chemical modification, it is necessary to investigate whether these chemical changes lead to cytotoxic effects. For this reason, in this study the modified cellulose was investigated in respect to their cytotoxic, genotoxic and mutagenic effects. These studies are important to evaluate the behavior of the regenerated cellulose for future in vivo applications.
Therefore, this study aimed to prepare regenerated cellulose scaffold, systematically characterize it, and evaluate its enzymatic degradation as well. In addition, the potential cytotoxic, genotoxic and mutagenic effects of these materials were evaluated.
Section snippets
Preparation of regenerated cellulose scaffolds
Pristine cellulose pulp (CP) and pristine regenerated cellulose scaffold (RCS), prepared by the viscose process, were supplied by Coopercell Ind. de Papel Celofane (São Paulo, Brazil). Cellulose was firstly immersed in 18% (w/v) NaOH solution at room temperature; subsequently the alkali cellulose was reacted with CS2 solution at 30 °C for 90 min leading to cellulose xanthate. H2SO4 solution was added regenerating cellulose. The formation of regenerated cellulose scaffold with controlled pore size
CP and RCS characterization
CP and RCS showed different morphologies due to the modification of hydrogen bond patterns and crystalline structure during regeneration process. Porous samples can be easily produced by adding a pore forming material (sodium sulfate salt) in the cellulose solution.
The original cellulose (CP) and the porous regenerated cellulose (RCS) are presented in photographs of Fig. 1a and b, respectively. The morphology of the samples was evaluated by using scanning electron microscopy (SEM), the
Conclusions
Regenerated cellulose scaffolds were prepared by viscose process. The samples exhibited structure compatible with the cellulose II, low crystallinity and a lower thermal stability. These requirements are important to ensure biodegradability of the scaffold. RCS also showed a slower in vitro degradation profile for samples containing lysozyme compared to samples containing Tr cellulase. In addition, RCS did not show cytotoxicity, mutagenic and genotoxic potential. Thus, we can conclude that the
Acknowledgements
The authors thank Brazilian agencies FAPESP, CAPES and CNPq for financial support. In addition, the company Coopercell Ind. de Papel Celofane (São Paulo, Brazil) for supplying the pristine regenerated cellulose and the LMA-IQ for FEG-SEM facilities.
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These authors contributed equally to this paper.