Research paperPhysico-chemical, mechanical and cytotoxicity characterizations of Laponite®/alginate nanocomposite
Introduction
Combining organic and inorganic biomaterials benefits from the strengths and advantages of both phases in one hybrid. Mixing silica-based biomaterials (clay minerals) as an inorganic compound with synthetic polymers or biopolymers (naturally-derived polymers) has been extensively studied in emerging advanced biological technologies (Chen and Xu, 2009, Chen and Yoon, 2005, Chrzanowski et al., 2010, Esumi et al., 1998, Gaharwar et al., 2011b, Gaharwar et al., 2011c, Gaharwar et al., 2011d, Gaharwar et al., 2012, Lewkowitz-Shpuntoff et al., 2009, Senillou et al., 1999, Wu et al., 2010). Clay polymer nanocomposites (CPN) have been used in medical diagnostic and therapeutic devices, biomedical imaging, regenerative medicine and controlled drug delivery devices, as well as some non-biological applications such as electronics, water waste treatment, reinforcing structural and coating materials (Chen and Xu, 2009, Chen and Yoon, 2005, Esumi et al., 1998, Gaharwar et al., 2011b, Gaharwar et al., 2011c, Gaharwar et al., 2011d, Gaharwar et al., 2012, Lewkowitz-Shpuntoff et al., 2009, Senillou et al., 1999, Wu et al., 2010). CPN have shown promising properties as biomaterials due to the enhanced surface interactions between polymer chains and silica groups present in clay mineral (Gaharwar et al., 2011d).
Smectite mineral family, such as Montmorillonite, Cloisite and Laponite, are the most commonly used clay minerals in various biomedical applications (Ray and Okamoto, 2003). This is due to the layered structure of smectite mineral, and thereby their high specific surface area, adsorptive capacity, surface reactivity, and cation exchange capacity (CEC) (Ray and Okamoto, 2003). In CPN, the degree of dispersion of layered silicate nanoparticles in polymer matrix depends on the level of clay mineral CEC and its specific surface area. CEC of clay mineral indicates its ability of absorbing and exchanging cations in their layered structure (Wu et al., 2010). Several studies blended smectite with different polymers to produce new biomaterials, for example, mixing clay with polyethylene oxide (PEO), polyacrylamide (PAM), polylactic acid (PLA), polylactide-co-glycolide acid (PLGA), poly-N-isopropylacrylamide (PNIPAM), polyurethanes (PU), polyethylene glycol (PEG) and some natural biopolymers like chitosan, gelatin and alginate (Gaharwar et al., 2011b, Gaharwar et al., 2011c, Gaharwar et al., 2011d, Gaharwar et al., 2012, Haraguchi and Takehisa, 2002, Haraguchi et al., 2006, Hawkins et al., 2009, Laftah et al., 2011, Li et al., 2009).
Laponite, Na0.7[(Mg5.5Li0.3)Si8O20(OH)4]0.7, is a synthetic smectite with similar chemical composition to the bioactive glasses (SiO2, Na2O, CaO, MgO, P2O5). Laponite is biocompatible, and non-toxic (free of organic and heavy metals contaminations), with nano-size layered structure material, which has been used as a model of silica-based nanoparticle in CPN studies (Gaharwar et al., 2011b, Gaharwar et al., 2011c, Gaharwar et al., 2011d, Gaharwar et al., 2012, Ruzicka and Zaccarelli, 2011, Schexnailder and Schmidt, 2009). It has been reported that incorporating silicate nanoparticles (Laponite) into polymers improves mechanical strength, modulates elasticity, stiffness and swelling, and enhances biological activities (e.g.; cell adhesion and proliferation) of the nanocomposite compared to the polymer alone (Gaharwar et al., 2011b, Gaharwar et al., 2011c, Gaharwar et al., 2011d, Gaharwar et al., 2012, Hu et al., 2004, Schexnailder et al., 2010, Shi et al., 2008, Yang et al., 2011, Zhang et al., 2009). Changes in mechanical properties depend on the Laponite composition and percentage, polymer/silicate nanoparticle interactions and the degree of cross-linking (both physical and covalent) within polymer chains and between silicate nanoparticles and polymer (Gaharwar et al., 2008a, Gaharwar et al., 2008b, Gaharwar et al., 2010a, Gaharwar et al., 2010b, Gaharwar et al., 2011a, Gaharwar et al., 2011b, Gaharwar et al., 2011c, Gaharwar et al., 2012). In regards to the biological activities of Laponite-based CPN, it has been shown that the presence of silicate nanoparticles enhances proliferation, differentiation and adhesion of cells seeded on the surface of CPN compared to the polymer alone (Gaharwar et al., 2011b, Gaharwar et al., 2012, Schexnailder et al., 2008, Schexnailder et al., 2010).
In the present study, sodium alginate, a natural polysaccharide, has been chosen to mix with Laponite due to alginate versatile functionality, gentle gelling kinetics, low cost, biocompatibility, low toxicity and environmental friendly nature (Luginbuehl et al., 2005). Sodium alginate has been widely used in biomedical applications mainly as a material for cell encapsulation because of its ability in maintaining cell viability within cross-linked gel (Trivedi et al., 2001). Alginate has also been used alone or in combination with other biomaterials such as gelatin, chitosan, polyvinyl alcohol (PVA) and non-organic particles like hydroxyapatite, magnesium aluminum silicate, tri-calcium phosphate and organosiloxane as biomaterial in various applications. For instance, mixing alginate with gelatin and chitosan was studied in bone, cartilage, heart and endovascular system grafts (Stancu et al., 2011, Wang et al., 2010). Combination of PVA (polyvinyl alcohol) and alginate has also been applied as wound dressing and drug delivery matrix (Shalumon et al., 2011).
Despite alginate has been used in many applications, its low mechanical strength is a main limitation for alginate-based materials to be used in load bearing applications. To overcome this problem, prior researchers blended fillers like hydroxyapatite, magnesium aluminum silicate, and tri-calcium phosphate to the alginate matrix, which resulted in improvement of alginate mechanical strength (Lawson et al., 2004, Lee et al., 2011, Weir et al., 2006). In addition to its inferior mechanical strength, it has been reported that alginate gel does not provide sufficient support for mammalian cell adhesion (Alsberg et al., 2001). Different methods were developed to improve cell adhesion on alginate, including surface coating with cell recognition sites like RGD (arginine-glycine-aspartate sequences) that favor cell adhesion to the surface of materials (Shin et al., 2004, Yu et al., 2009). Other researchers suggested that incorporating silica-based particles into polymers such as polyethylene oxide, ethylene vinyl acetate and chitosan, resulted in improved cell adhesion, proliferation and differentiation in comparison to the polymer alone (Gaharwar et al., 2011d, Jin et al., 2009). These studies demonstrated that clay minerals change polymer properties such as hydration, dissolution and mechanical properties, resulting in cell–polymer interaction that supports cell adhesion.
To address low mechanical strength and cell adhesion capability of alginate, Laponite/alginate (Lap/Alg) nanocomposites with different ratio of Laponite were developed in this study. The study therefore aimed to produce a nanocomposite with enhanced mechanical properties and improved cell adhesion. It was envisaged that this nanocomposite is to be injected into damaged cartilage and/or bone sites and set in-situ by cationic cross-linking of alginate chains. The physico-chemical and mechanical properties of the Lap/Alg nanocomposite as well as its cellular interaction and cytotoxicity were investigated.
Section snippets
Lap/Alg nanocomposite preparation
4% sodium alginate, low viscosity, 100–300 cP, (Sigma-Aldrich Pty. Ltd) was dispersed in 100 ml of deionized water. Laponite® XLG powder, layered synthetic clay mineral with the thickness of ca. 1 nm and a diameter of ca. 30 nm (Rockwood additives) was added to the freshly made 4% alginate solution under vigorous stirring process. Nanocomposites with the Laponite:alginate mass ratios of (20:80), (50:50) and (80:20) were prepared. The mixture was then filled in a 12 well-plate and set and casted
Physico-chemical properties and Young modulus of Lap/Alg nanocomposite
Fig. 1 illustrates the Young modulus of hydrated alginate and Lap/Alg nanocomposites, which increased by elevating the Laponite content in nanocomposites. The Young modulus of the 20:80 nanocomposite (Laponite:alginate ratio) was 175 kPa, which increased to 305 kPa and 420 kPa in 50:50 and 80:20, respectively. In all nanocomposites, Young modulus was significantly higher than alginate alone (p < 0.05). The compressive modulus of nanocomposites in the present study found to be higher than those
Conclusions
Depending on the biomedical application, the prepared Laponite/alginate nanocomposites could be used as a potential biomaterial with the mechanical and rheological properties that can be modulated by changing the ratio between Laponite and alginate contents. The study showed that combining Laponite clay mineral with alginate improved alginate properties, including compressive strength, water uptake, and viscosity without hindering the injectability of materials. Although the surface of prepared
Acknowledgments
The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy and Microanalysis, University of Sydney. The authors would like to thank Mrs. Judy Ching Yee Loo from the Faculty of Pharmacy and Mr. Trevor Shearing from the Faculty of Engineering at the University of Sydney for their technical supports throughout this study.
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