Electrospinning is a well-established technique widely used for the fabrication of micro- and nanofibrous mats for numerous applications, including for the biomedical field, e.g. drug delivery and tissue engineering scaffolds. The principle at the basis of the electrospinning process is the application of a high voltage at the tip of a needle containing a polymer solution, suspension, blend or melt. During the time of flight the solvent evaporates from the charged solution and the obtained fibers are collected on the grounded target. The electrospinning process and the characteristics of the obtained electrospun mats are affected by several parameters. They could be grouped in three categories: solution parameters, process parameters and environmental parameters [1
In the tissue engineering field, the versatility of this scaffold fabrication technique is proven by the increasing number of publications during the last years, by the integrated use of electrospinning with other scaffold fabrication techniques and by the development of a high number of different electrospinning setups [1
]. A huge number and variety of polymers can be processed by electrospinning, but often the use of organic toxic solvents is required. Recently, the introduction of the “Green Electrospinning” concept [4
] and the awareness about the disadvantages related to the use of toxic solvents, as environmental impact, safety related issues for the lab workers and the possible presence of residuals of toxic solvents in the electrospun mats, have raised the interest in the use of less or not toxic solvents (benign solvents) for electrospinning [5
Poly(epsilon-caprolactone) (PCL) is a biodegradable linear aliphatic polyester which is widely used for tissue engineering, biomedical, food and other industrial applications [7
]. PCL usually exhibits a low degradation rate in aqueous solution, with non-toxic products of degradation. Even if its hydrophobicity cannot promote and allow cell adhesion, it is possible to post-treat the sample in order to modify its surface without affecting the fibrous structure, and different types of treatments are already reported in literature [10
]. It is also possible to blend PCL with other polymers in order to modulate its properties [13
Since the beginning of the spreading of the electrospinning technique, PCL, other polyesters and their blends have been processed by this method [16
]. PCL is soluble in tetrahydrofuran, chloroform, methylene chloride, carbon tetrachloride, benzene, toluene, cyclohexanone dihydropyran and 2-nitropropane and only partially soluble in acetone, 2-butanone, ethyl acetate, acetonitrile and dimethyl fumarate [18
]. For the electrospinning process, it is common to solve PCL using mixtures of solvents and co-solvents, like chloroform/methanol, methylene chloride/methanol, methylene chloride/N,N-dimethylformamide (DMF), methylene chloride/toluene or tetrahydrofuran/DMF [20
]. Unfortunately most of these solvents suitable for electrospinning are toxic and harmful, for this reason, recently several research works have focused on the use of less toxic and harmful solvents for electrospinning, i.e.
acetic acid, formic acid and acetone [5
]. These studies have demonstrated the increased focus on less harmful solvents for electrospinning but they have also highlighted that most of these solvents are not directly suitable for electrospinning, requiring a longer and more accurate optimization of the process.
For tissue engineering applications, considering the importance of mimicking the native structure and function of the extracellular matrix (ECM), the electrospinning technique is particularly relevant for the obtainment of nanosized fibers [1
]. As already reported in literature by Soliman et al.
], it is possible to obtain PCL nano- and microfibers by just regulating and adjusting the polymeric solution concentration without changing solvents, however in such cases the solvent is usually a mixture of chloroform and methanol. Recently, other studies have considered the fabrication of PCL nanofibers using less harmful solvents, like formic acid. In fact, formic acid is relevant for the reduction of the fiber diameter, as already reported by Van der Schueren et al.
], who showed that the increase of the amount of formic acid in the electrospinning solution leads to a decrease in the average fiber diameter.
The relevance of the presence of macroporosity in electrospun fiber mats has been highlighted by the need of pores able to allow cell infiltration inside the electrospun mats and, considering the density of the fibrous structure, often cells could only adhere on the surface without penetrating inside the mesh [2
]. The dimension and the interconnectivity of the porosity are crucial elements for tissue engineering scaffolds and they are also related to the cell type and the target tissue [27
The electrospinning technique has been widely used to pursue a biomimetic approach to obtain composite structures [28
] in order to reproduce the composition of native tissue. In fact the suitability of the electrospinning of suspensions containing inorganic phases in the polymeric solutions has been already investigated for bone tissue engineering applications [29
]. In particular, for this goal, the use of bioactive glass (BG) particles is relevant to promote mineralization of the constructs and also because of the BG bioactivity, and its effect on osteogenesis and angiogenesis processes [31
In this framework, the aim of the present work has been the optimization of the electrospinning of poly(epsilon-caprolactone) (PCL) using acetic acid and a mixture of acetic acid and formic acid as solvents, demonstrating the feasibility of producing bead-free micro- and nanofibers with the use of such benign solvents. Beyond the electrospinning process optimization, the novelty of the present work resides in the evaluation of the suitability of these solvents for the fabrication of patterned mats, which allows the formation of macroporosities relevant for improving cell infiltration inside the electrospun mats. In addition, the versatility of the solvents for the fabrication of composite fibers, obtained with the addition of bioactive glass particles inside the polymeric solution, was considered in order to obtain organic-inorganic composite electrospun mats suitable for bone tissue engineering applications.
3. Experimental Section
Electrospun mats were obtained from PCL (80 kDa, Sigma Aldrich, Munich, Germany) solutions. Acetic acid (AA, VWR, Darmstadt, Germany) and formic acid (FA, VWR, Darmstadt, Germany) were used as solvents.
The details of each solution (solvent and polymer concentration) are reported in Table 3
. The solutions of PCL in acetic acid were stirred overnight and then were put in an ultra-sound bath for 1 hour. For the fabrication of the composite electrospun mats, commercially available BG particles (Schott Vitryxx®, size 2 μm, Schott AG, Mainz, Germany) were homogeneously dispersed (30 wt % respect to PCL) in the polymer solution and stirred for 10 minutes. The PCL solution used for PCL-BG samples was the solution containing 20% w
of PCL in acetic acid.
3.2. Electrospinning Process
The optimization of the electrospinning parameters was performed and different solution concentrations and solvents systems were evaluated, as summarized in Table 3
. Electrospinning was performed using a commercially available setup (Starter Kit 40KV Web, Linari Engineering srl, Valpiana (GR), Italy). The optimized electrospinning parameters for the solution of 20% w
of PCL were an applied voltage of 15 kV, a needle-target distance of 11 cm, while the polymeric solution was fed with a flow rate of 0.4 mL/h.
Samples morphology was assessed by SEM analysis (FE-SEM-EDS (Auriga 0750, Zeiss, Jena, Germany)). Samples were sputtered with gold before SEM analysis using a Sputter Coater (Q150T, Quorum Technologies, Darmstadt, Germany). Fiber average diameters were calculated using ImageJ (NIH, Bethesda, MD, USA), after the measurement of 50 fibers from each sample.
FTIR spectra of selected samples were obtained using an FTIR spectrometer (Nicolet 6700, Thermo Scientific, Schwerte, Germany) in attenuated total reflectance mode (ATR). For the analysis, 32 spectral scans at a resolution of 4 cm−1 were repeated over the wavenumber range 4000–550 cm−1. The used window material was CsI.
The mechanical characterization of selected fibrous mats was performed by uniaxial tensile strength test, by using a universal testing machine (Zugfestigkeitsprüfmaschine Frank, K. Frank GmbH, Mannheim, Germany) at room temperature. The measurements were carried out at a crosshead speed of 10 mm/min by using a 50 N load cell. In order to handle correctly the electrospun mats, avoiding the application of any pretension on the samples before the mechanical tests, the use of a suitable paper square framework was necessary. The samples were cut in rectangular shape of 5 mm wide and 4 cm long and the internal length of the paper framework was 2 cm.
The acellular bioactivity of the electrospun composite samples containing BG was evaluated by the immersion of the samples in a simulated body fluid (SBF) solution, according to the protocol reported in ref. [41
], for 1, 4 and 7 days. After the immersion, the samples were characterized by SEM, EDX and ATR-FTIR analyses. Samples of neat PCL electrospun mats were used as control. The bioactivity of the samples was related to the formation of hydroxyapatite phase on the surface of samples, upon immersion in SBF [41