Solvent-dependent properties of electrospun fibrous composites for bone tissue regeneration
Introduction
Calcium phosphates have been used as bone graft substitutes for many years and, more recently, have been sought as bone tissue engineering scaffolds. The two most commonly investigated bioceramics are β-tricalcium phosphate (β-TCP) (Ca3(PO4)2) and synthetic hydroxyapatite (HA) (Ca10(PO4)6(OH)2). These materials are biocompatible, osteoconductive, bioactive and, depending upon the composition, biodegradable. Several studies have also reported that calcium phosphate ceramics have osteoinductive properties [1], [2], [3]. In combination with bone marrow derived mesenchymal stem cells (MSC), calcium phosphate ceramics provide the necessary cues for stem-cell-induced bone tissue formation [4], [5], [6]. Clinically, however, ceramics have had limited use because of their brittleness and difficulty in shaping [7]. Therefore, biodegradable polymer-bioceramic composites have been sought as an alternative form to using calcium phosphates alone. The most commonly used polymers for composites are poly(l-lactic acid) (PLLA), poly(lactic-glycolic acid) (PLGA) and polycaprolactone (PCL).
There are various methods of fabricating composite scaffolds for bone tissue engineering, such as solvent casting [8], gas foaming [9], phase separation [10] and electrospinning [11]. In this work, electrospinning was used for scaffold fabrication. This method of scaffold fabrication has gained importance in the field of tissue engineering, mainly because of the high surface to volume ratio of the scaffolds, by the creation of ultrafine and continuous micron to nanofiber structures, which is important for improving the level of protein adsorption and subsequent cell attachment [12]. Recently, researchers have begun to investigate the fabrication of composite electrospun mats. However, there are only a few published reports with limited characterization [11], [13], [14], [15], [16]. In addition, the mats in these studies have pore/void spaces below the dimension of the size of a cell, which results in poor cell infiltration and could limit tissue in-growth in vivo. These previous studies also have limited characterization with respect to the polymer and ceramic phases and their molecular interaction.
In this study, composite scaffolds of PCL and 20%HA/80% β-TCP were produced by electrospinning. The goal was to fabricate mechanically flexible composite scaffolds with uniform fiber morphologies, relatively large pore sizes for cell infiltration and bone tissue in-growth, a maximum concentration of ceramic to achieve improved bioactivity [17] and a homogeneous dispersion of the ceramic in the fibers for improved molecular interaction and mechanical properties [18]. Previous studies have demonstrated the potential of biphasic compositions of HA and β-TCP ceramics for bone tissue engineering applications. The composition 20%HA/80% β-TCP was chosen for this study because this composition has demonstrated favorable results both in vivo [19], [20] and in vitro [19], [21]. The rate of bone tissue formation has been shown to be greater on this composition compared with HA or TCP alone. In addition, at the cellular level, 20%HA/80% β-TCP has been shown to stimulate the osteogenic differentiation of human MSC [19]. HA resorbs very slowly and β-TCP resorbs relatively quickly [22]. This biphasic composition may achieve an optimum balance in stability vs degradation, which, in turn, promotes bone tissue formation [19], [23].
Therefore, this study fabricated and for the first time investigated an electrospun composite consisting of 20% HA/80% β-TCP and PCL as a potential scaffold for bone tissue engineering applications. Solvent, solvent combinations and varying weight percentages of the ceramic for the preparation of the polymer–ceramic composites were investigated. An expanded protocol of physical characterization of the composite was performed using scanning electron microscopy (SEM), X-ray, thermal and mechanical analyses. An in vitro study of cell growth and morphology on these composites was also investigated using MSC derived from adult bone marrow as a potential cell source for a bone tissue engineering strategy.
Section snippets
Materials
Poly (ε-caprolactone) (PCL) with MW 80,000 was purchased from Sigma Aldrich, Inc. HA and β-TCP with average particle size 100 nm were obtained from Berkeley Advanced Biomaterials, Inc. (Berkeley, CA). The solvents used for electrospinning were methylene chloride (MC) (density 1.32 g cm−3, boiling point 39.75 °C) and dimethylformamide (DMF) (density 0.944 g cm−3, boiling point 153 °C) from Fisher Scientific, Inc.
Scaffold fabrication
The scaffolds were fabricated using the electrospinning process. The basic principle behind
Scaffold fabrication and characterization
Composite–MC mats were fabricated using up to 50 wt.% of the ceramic. Higher concentrations of ceramic were not attempted, because the mats became too weak to handle, and the viscosity of the solution became too high for electrospinning. The SEM results showed a bimodal distribution of fiber diameters with sub-micron-sized fibers forming a web-like network in-between larger diameter fibers using this method of fabrication. These structures can be seen in Fig. 1a and b (30% ceramic) and Fig. 1d
Discussion
Electrospinning, though typically used for producing nano-sized fibrous mats, can also be used for forming micron-sized fibers by controlling the parameters used for the electrospinning process. A major drawback of electrospinning is limited control of inter-fiber spacing [28], where 100-nm-dia. fibers can yield a mean pore size at <10 nm, a relative density of 80% [29]. Since one of the objectives of this study was to produce larger pore sizes, the procedures used were designed to produce
Conclusion
This study fabricated and extensively characterized an electrospun composite consisting of 20% HA/80% β-TCP and PCL as a potential scaffold for bone tissue engineering applications. Composite scaffolds with uniform fiber morphologies and uniform ceramic dispersions were successfully created using the electrospinning process with a two-solvent system. The electrospinning process, in which PCL was dissolved in MC, had different properties from that of the process where a combination of MC (80%)
Acknowledgement
The authors would like to thank support from NSF PECASE CBET-0238787 (Arinzeh).
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