Chitosan(PEO)/silica hybrid nanofibers as a potential biomaterial for bone regeneration

Abstract

New hybrid nanofibers prepared with chitosan (CTS), containing a total amount of polyethylene oxide (PEO) down to 3.6 wt.%, and silica precursors were produced by electrospinning. The solution of modified sol–gel particles contained tetraethoxysilane (TEOS) and the organosilane 3-glycidyloxypropyltriethoxysilane (GPTEOS). This is rending stable solution toward gelation and contributing in covalent bonding with chitosan. The fibers encompass advantages of biocompatible polymer template silicate components to form self-assembled core–shell structure of the polymer CTS/PEO encapsulated by the silica. Potential applicability of this hybrid material to bone tissue engineering was studied examining its cellular compatibility and bioactivity. The nanofiber matrices were proved cytocompatible when seeded with bone-forming 7F2-cells, promoting attachment and proliferation over 7 days. These found to enhance a fast apatite formation by incorporation of Ca²⁺ ions and subsequent immersion in modified simulated body fluid (m-SBF). The tunable properties of these hybrid nanofibers can find applications as active biomaterials in bone repair and regeneration.

Introduction

Sol–gel derived hybrid silica glass nanocomposite materials and nanofibers have been recently introduced as scaffolding matrices for bone–tissue regeneration (Heinemann et al., 2009, Heinemann et al., 2011, Kim et al., 2006, Mahony et al., 2010, Poologasundarampillai et al., 2010, Seol et al., 2010). These organic–inorganic nanocomposite materials combine both counterparts in one material, advantaging the flexibility and good mold ability of the organic part, heat-stability, high strength and chemical resistance of the inorganic part. Moreover, coating ability of apatite on ceramics has been known for a long time (Kokubo, 1991) and has been tested on silica glasses of various alkoxide systems (Kim et al., 2006, Poologasundarampillai et al., 2010, Toskas et al., 2011b). The silica hybrid materials favor cell attachment and are not cytotoxic as demonstrated by culture of mesenchymal stem cells (MSCs) on a silica–gelatin hybrid (Mahony et al., 2010), on silica–collagen composites (Heinemann et al., 2009) and that of an osteosarcoma cell line SaOs-2 on a poly(γ-glutamic acid)/silica hybrid (Poologasundarampillai et al., 2010).

Chitosan (CTS) a natural cationic polymer offers unique properties such as biologically renewable, biodegradable, biocompatible, non-antigenic, non-toxic and biofunctional. It is promoting cell adhesion, proliferation and differentiation and evokes a minimal foreign body reaction on implantation (Muzzarelli, 2009). Chitosan with silicate hybrids were synthesized with glycidoxypropyltrimethoxy silane (GPTMS), whose epoxy groups are considered to react with the amino groups of chitosan. The cross-linking density was around 80% regardless of the amount of silane (Muzzarelli, 2011). The values of the mechanical parameters indicated that significant stiffening of the hybrids was obtained upon addition of the silane while full flexibility was retained. In addition, adhesion and proliferation of the MG63 osteoblast cells cultured on the hybrid surface were improved compared to those on the pure chitosan membrane regardless of the silane concentration (Muzzarelli, 2011, Shirosaki et al., 2005, Shirosaki et al., 2009a). MC3T3-E1 cells also showed comparable viability on sol–gel derived silica xerogel/chitosan hybrid coatings onto alkali-treated titanium, proving their potential to be used as bioactive coating materials in hard tissue engineering (Jun et al., 2010). Moreover chitosan was found to improve structural integrity of PEO cross-linked by silicate nanoparticles (Laponite) films and to enhance the formation of a mineralized extracellular matrix and the differentiation of MC3T3-E1 preosteoblast cells (Gaharwar, Schexnailder, Jin, Wu, & Schmidt, 2010).

Glass ceramic silica (SiO₂) electrospun nanofibers have received significant attention due to their multifunctional properties in biomedical fields (Kim et al., 2006, Seol et al., 2010, Sridhar et al., 2012). There are two main methods of preparing silica electrospun nanofibers via solutions (spin dopes): the first is by the direct spinning from aged sol–gel solutions containing alkoxide precursors; the second involves carrying polymers and is more advantageous as it can be easily adjusted, generating nanofibers of controllable size and uniformity. As alkoxide precursor, tetraethoxysilane (TEOS) and tetramethoxysilane (TMOS) are commonly used (Choi et al., 2003, Shao et al., 2003). As carrying co-electrospun homopolymer polyvinyl pyrrolidone (PVP), polyvinylidene fluoride (PVDF), nylon-6 and polyvinyl alcohol (PVA) are common studied polymers (Gaharwar et al., 2010, Kim et al., 2010, Liu et al., 2008, Shao et al., 2003). Recently, new types of organic–inorganic silica nanofibrous (SiO₂) composite membranes developed based on one of the most frequently used non-toxic and biocompatible polymers, polyethylene oxide (PEO) (Toskas et al., 2011b). A new organically modified alkoxide sol–gel solution was used containing a mixture of tetraethoxysilane (TEOS) and 3-glycidyloxypropyltriethoxysilane (GPTEOS). Organosilanes such as GPTEOS are having the advantage of bearing organic and inorganic functionalities in the one molecule which provide the ability to bond to organic chains and inorganic moieties accordingly. Moreover, major advantage of this combined alkoxide sol–gel precursor is to increase the stability of GPTEOS toward gelation up to several months (Mahltig, Fiedler, Fischer, & Simon, 2010). It is also suggested that organosilanes as γ-glycidoxypropyltrimethoxy silane (GPTMS) slow condensation reaction rate and enhance the polymer–silica compatibility (Liu, Su, & Lai, 2004).

Chitosan (CTS) nanofibers via electrospinning were first obtained from neat trifluoroacetic acid (TFA) at 7 wt.% (Ohkawa, Cha, Kim, Nishida, & Yamamoto, 2004). But the more usual method involves the use of polyethylene oxide (PEO) as co-blending polymer, in order to spin chitosan at higher polymer concentrations (Bhattarai et al., 2005, Klossner et al., 2008, Toskas et al., 2009). Combining the unique collective properties of both materials, the fabrication of composite organic–inorganic CTS/SiO₂ nanofibers via electrospinning is of great interest. Introduction of silica into biomaterials was early proved to increase its oxygen permeability, biocompatibility, and biodegradability (Suzuki and Mizushima, 1997, Tian et al., 1997). In this work, a controllable process of creating CTS(PEO)/SiO₂ nanofibers is presented; the effect of varying the polymer to silica precursor weight ratio is examined and correlated to the structural properties of the nanofibers. Subsequently, the ability of these hybrid nanofibers to favor cell attachment of 7F2 osteoblasts is tested and also their capability to modify by incorporating calcium ions resulting in bioactive hydroxyl carbonate apatite (HCA) crystal formation were in parallel examined.