Fabrication, characterization and in vitro biocompatibility of electrospun hydroxyethyl cellulose/poly (vinyl) alcohol nanofibrous composite biomaterial for bone tissue engineering

Highlights

• Fabrication of hydroxyethyl cellulose and polyvinyl alcohol nanofibers with different weight concentrations.

• Thermo-mechanical and chemical properties of electrospun nanofibers.

• Biocompatibility of fabricated nanofibers using human osteosarcoma cells (U2OS).

Abstract

Development of novel scaffold materials that mimic the extracellular matrix, architecturally and functionally, is becoming highly important to meet the demands of the advances in bone tissue engineering. This paper reports, the fabrication of natural polymer cellulose derived hydroxyethyl cellulose (HEC) based nanostructured scaffolds with uniform fiber morphology through electrospinning. Poly (vinyl alcohol) (PVA) was used as an ionic solvent for supporting the electrospinning of HEC. Scanning electron microscopy and ImageJ analysis revealed the formation of non-woven nanofibers with well-defined porous architecture. The interactions between HEC and PVA in the electrospun nanofibers were studied by differential scanning calorimetry, X-ray diffraction, dynamic mechanical analysis thermo-gravimetric analysis; Fourier transform-infrared spectroscopy, X-ray photoelectron spectroscopy and tensile test. The mechanical properties of scaffolds were significantly altered with different ratios of HEC/PVA. Further, the biocompatibility of HEC/PVA scaffolds was evaluated using human osteosarcoma cells. The SEM images revealed favorable cells attachment and spreading on the nanofibrous scaffolds and MTS assay showed increased cell proliferation after different time periods. Thus, these results indicate that HEC based nanofibrous scaffolds will be a promising candidate for bone tissue engineering.

Introduction

Tissue engineering is an interdisciplinary field that combines the knowledge of cells, engineering materials, and suitable biochemical factors for development of a suitable artificial cellular scaffold to maintain, or regenerate the damaged tissues (Agarwal et al., 2009, Rodriguez et al., 2011, Wenjing et al., 2014). The scaffold is a support that mimics the extracellular matrix (ECM) and serves as a temporary skeleton for cell attachment proliferation and differentiation to reconstruct new organs and tissues. An ideal scaffold for bone tissue regeneration desires appropriate surface chemistry and microstructure with good mechanical properties, biocompatibility and bioactivity (Rodriguez et al., 2011; Wenjing and Jiashu, 2014). A perfect scaffold involves the selection of suitable material which is biocompatible, biodegradable, as well as nontoxic to the cells both in the original and degraded forms (Martins et al., 2007). In addition to the selection of material, another important requirement is a suitable method to fabricate the scaffold with micro to nanoscale topographical features. Various techniques gained interest in the development of bone scaffolds from numerous compositions of different polymeric or non-polymeric materials. Among them, electrospinning has attracted much attention for the fabrication of nanofibrous scaffolds. The inherent non-woven nature of the electrospun nanofibers can serve as an ideal scaffold to mimic the extracellular matrix (ECM) for cell attachment and nutrient transportation owing to its high porosity and large surface-area-to-volume ratio (Jianga et al., 2015, Zhang et al., 2005a).

For the past few decades many natural and synthetic polymers as well as their blends have been widely used for bone tissue engineering. Collagen, chitosan and chitin are the most widely used natural polymer. Due to the structural and functional similarity of collagen to natural bone extra cellular matrix it has attract more attention as a bone scaffold (Zhang et al., 2005b, Sheeny et al., 2014). Due to the high cost of collagen, it was substituted by gelatin based materials which possess excellent biodegradability and non-antigenicity but also have lower mechanical strength (Zhang et al., 2005c, Poinern et al., 2009). When processed into nano-scaffolds, natural polymers were also found to lack the appropriate mechanical strength for applications in bone tissue engineering (Yang et al., 2008, Kanungo et al., 2008, Cen et al., 2008). Polyesters [e.g., PLLA (polylactides), PLGA (polyglycolide), and PCL (polycaprolactone)] are a very common group of synthetic polymers that are used to construct biodegradable nanofibrous materials for bone tissue engineering (Heydarkhan-Hagvall et al., 2008, Lu et al., 2014, Zhang et al., 2006). These biomaterials can be used to form porous scaffolds with very high tensile properties, which are suitable candidates for bone tissue regeneration. However, their degradation rates are not convenient and the degradation products are acidic in nature, moreover these materials require toxic chemicals as a solvent (Goonoo et al., 2013).

Polysaccharide chains mainly composed of D-glucose unit named as cellobiose and it is the main constituent of cellulose. Cellulose consists of two glucose residues linked via a β-1,4 glycosidic bond, which make it as one excellent materials for tissue engineering applications (Ding et al., 2014). In last few years the cellulose based material specially cellulose nanocrystals and nanofibrilled cellulose have widely investigated for tissue engineering applications due to their excellent biocompatibility, biodegradability and low cytotoxicity to a range of animal and human cell types (Azizi Samir et al., 2005, Kmmerer et al., 2011, Kovacs et al., 2010). Hydroxyethyl cellulose (HEC) is one of the leading cellulose derivative and is extensively used in the pharmaceutical industry (Domingues et al., 2014). It is non-ionic, water soluble polymer and has the β-glucose linkage, which makes it a suitable candidate for tissue engineering applications. PVA has gained acceptance as a scaffold supporting material for tissue engineering applications, due to its biocompatibility, biodegradability and chemical resistance properties. It provides mechanical stability and flexibility to the conventional scaffolds made of natural polymers. It is frequently employed electrospun polymer in drug delivery and tissue engineering (Krumora et al., 2000). Our research group recently published HEC/PVA based electrospun nanofibers biomaterials for skin tissue engineering application (Zulkifli et al., 2014). That paper reported the use of low molecular weight PVA (~95,000) and very high molecular weight HEC (~25,0000). In our recent articles we have reported the calcium phosphate functionalized HEC/PVA nanofibrous scaffolds (Chahal et al., 2014, Chahal et al., 2015)

In this work, we report the fabrication of HEC (low molecular weight) based electrospun biomaterials with different PVA (high molecular weight) concentrations. Electrospinning of HEC polymer solution is very difficult, due to its non-ionic nature; hence PVA is used as the polymer additive to produce electrospun nanofibers. The morphology of the resulting nanofibers was analyzed by scanning electron microscopy (SEM). The enhancement of the thermos-mechanical properties of electrospun HEC/PVA nonwoven mats was demonstrated by thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). Finally, the biocompatibility of prepared nanofibers was evaluated using human osteosarcoma cells. The cells morphology and viability on the HEC/PVA nanofibers mats were evaluated by SEM, MTS essay.