Structure of poly(lactic-acid) PLA nanofibers scaffolds prepared by electrospinning

The structural properties of poly(lactic-acid) PLA nanofiber scaffolds prepared by electrospinning have been correlated with their process condition. The influence of the electrospinning processing parameters on structure including fiber orientation, take-up velocity and post-thermal treatment was analyzed. The structure and the properties of the scaffolds were studied by x-ray diffraction (XRD), atomic force microscope (AFM), scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), and differential scanning calorimetry (DSC). The existence of crystallinity into the nanofibres of PLA was found. The careful observation by HRTEM shows an amorphous core and a semicrystalline shell structure (supramolecular), and the surface of nanofiber observed by AFM shows a laminate periodic along the main axis of the fiber. These observations will be useful in understanding the structure-property relationships of oriented nanofibre scaffolds for medical or biological applications.


Introduction
The use of scaffolds and porous materials produced by several techniques are more common nowadays for tissue engineering [1]. A global tendency is the use of biodegradable biomaterials, similar to native tissues, composed by fibers like collagen, which might promote the cell growth and the extracellular matrix generation [2][3][4]. In order to achieve this goal, several techniques have been used to obtain scaffolds such as drawing, template synthesis, phase separation, self-assembly, biocomponent extrusion and electrospinning among others. Among the several techniques to produce scaffolds, electrospinning is one of the most versatile alternatives for controlling the structural parameters of fibrous scaffolds such as diameter, texture, porosity and fiber orientation [4][5][6][7]. 1 * To whom any correspondence should be addressed. An interesting work about the effect of crystallinity of PLA microfiber by electrical and centrifugal field on mechanical responses was described by Liao et al. [8]. Lim and Tan reported that electrospun polycaprolactone (PCL) nanofibers with smaller diameters had a higher degree of molecular orientation, crystallinity and mechanical properties [9]. In other paper, Tan and Lim reported the effect of annealing on electrospun poly(L-lactic acid) (PLLA) about crystalline structure after electrospinning [10]. They concluded that annealing was found to increase the Young's modulus of the nanofiber due to the increase in crystallinity and the change in morphology. It was clear that the fiber morphology affects the mechanical response of these materials. Wong et al. deduced an enhancement of molecular orientation and an abrupt shift in the elastic modulus of electrospun PCL occurred at around 700 nm in diameter [11].
Naraghi et al. studied the effect of diameter of polyacrylonitrile (PAN) nanofibers about the improvement of molecular orientation and mechanical properties [12]. Their results pointed out that molecular orientation is created at the late stages of the electrospinning process when the polymer jet viscosity increases and bending instabilities take place. Baji et al. also reported the influence of electrospinning parameters on the morphology and microstructure of several nanofibers (PLA, PCL, polyoxymethylene, nylon, cellulose acetate and other materials) and, in consequence, on their mechanical properties [13]. Yoshioka et al. studied the structure of polyethylene (PE) fibers by transmission electron microscopy (TEM) and the fiber structure was related to the fiber diameter. Therefore, they proposed three different structural models 1) the random-oriented crystalline 2) the shish-kebab and 3) the fibrillar structure [14].
Inai et al. have carried out tensile tests of single electrospun PLLA nanofibers collected in a rotating disc at different take-up velocities (tangential velocity produced by the rotating drum or disc) and found an improvement on the mechanical properties. Also, they reported the effects of the conductivity of the solution and the polymer concentration on the structure of PLLA electrospun [15].
Arinstein et al. suggested that nanofibers have a supramolecular structure composed by different phases [16]. Cicero et al [17] studied fibers of PLA produced by two-step melt-spinning. They found that structural properties can be widely manipulated through a combination of draw ratio and temperature. Using AFM, the fiber morphology as found to be highly fibrillar.
In our perspective there are fundamental issues about the internal fiber structure that must be taken into account such as the hierarchical structure. It has been suggested [16][17][18], but not clearly proven, that electrospun nanofibers can be visualized as composite materials, because they likely present an amorphous core and a supramolecular (semicrystalline) shell.
The internal structure and morphology of nanofibers of PLA has not been determined with enough precision because the nanometer dimensions. This work focuses on the effects of the velocity take-up and annealing on the microstructure of nanofibers of PLA. These experimental studies about microstructure of nanofiber of PLA and scaffolds structures was supported by a plausible inference of the experimental evidence from X-ray diffraction (XRD), differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM).

Materials and methods
Poly(lactid acid) of Mw 230000 (NatureWorks 2002D LLC, MN USA) was dissolved in 2, 2, 2trifluoroethanol (Sigma Aldrich) to obtain a 16 % w/v of PLA solution, using magnetic stirring. The PLA solution was electrospun to obtain both random and uniaxial oriented nanofiber scaffolds by using two different types of collector systems separately. The electrospinning process was performed at room conditions, at a flow rate of 0.6 ml/h and at an applied voltage of 15kV (Power supply Spellman, USA) and a digitally controlled pump (KD Scientific, USA). The collector for the random scaffolds was a 10 cm square aluminum plate. The collector to obtain oriented nanofibers is an aluminum cylinder custom-designed that rotate at a predetermined velocity in the range of 1 to 10000 RPM, providing tangential velocities (take-up velocities) ranging between 1 to 3142.6 m/min (52.36 . Subsequently, the electrospun scaffolds were kept in desiccators at room temperature. Some scaffolds were annealed at 80 °C during 10 hours (Scorpion Scientific, A-50980 oven). The annealing temperature was selected based on the crystallization temperature of as-spun aligned nanofibers (around 70°C). In general, annealing takes place at any temperature between the crystallization and the melting point.
The scaffolds structure was analyzed by X-ray diffraction, XRD, (Siemmens D-500, Cu Kα radiation; λ=1.5406 Å), differential scanning calorimetry, DSC, (2910, TA Instruments), scanning electron microscopy, SEM, (JEOL-JSM-7600F), atomic force microscopy, AFM, (Nanoscope III, Digital Instruments), and high resolution transmission electronic microscopy, HRTEM, (JEOL-2100f). ImageJ software was used to measure the mean diameter of the nanofibres and the orientation of the uniaxially aligned nanofibers (ANs). SEM images (10000 X) at two different locations of each sample were obtained. The diameters of the continuous fibers located at the foreground plane of the micrograph were measured. For the statistical analysis, at least eight points per nanofibre were selected to measure the diameters. The names and abbreviations of the electrospun fibers are shown in the Table 1.
The internal transverse structure of the oriented nanofibres was observed by HRTEM (JEOL-2100f). The samples were embedded in an epoxy resin Epon-812 (Microscopy ScienceR) and cured for 3 days, to impede the fibers from flexing during the cutting process, which was carried out in an Ultracryo Microtome RMCR model MT-7000. A conventional cutting pyramid was prepared, and fine sections (80-nm-thick) were obtained by means of a diamond blade (DIAMONTR) at room temperature, as the Tg for PLA is 60 °C. To obtain the proper contrast for the HRTEM observations, the samples were placed in a chamber under Ru04 vapour for 4 h. The surface of the ANs was observed by AFM (Nanoscope III, Digital Instruments) with a tip model TESP (Si) in the frequency range of 325-382 kHz (Veecoprobes). All images were taken in tapping mode and presented as height and phase images. The samples, 5x5 mm 2 , were cut and fixed at the standard AFM sample holder (metal disc). To avoid displacement and mechanical vibration of the nanofibres during the tip movement, a group of a few ANs was approached, and the AFM scan rate was kept below 0.3 Hz.

Characterization
The purpose of the characterization was to determine the structure and morphology of the PLA nanofibers and scaffold.

External morphology of electrospun nanofibers.
A set of SEM micrographs of PLA electrospun scaffolds is shown in the Figure 1. The degree of orientation of the AN nanofibers was analyzed at two take-up collection velocities; 1100 m/min (AN 1100 m/min) and 1217 m/min (AN 1217 m/min). The Figure 1A and 1B show that almost all nanofibers are aligned along the tangential direction of the cylindrical rotating collector. Both the diameter and degree of alignment of the nanofibers were measured by mean of the software: Image J. The average deviation angle of alignment was around +/-3 degrees. Therefore, it could be assumed that the nanofibers are uniaxially oriented. The average diameter of AN 1100 m/min nanofibers were 643.8 +/-50 nm, they showed a high degree of nanofiber orientation ( Figure 1A) with smooth and homogeneous surfaces ( Figure 1B). Randomly oriented nanofibers RON scaffolds had a greater average diameter: 992 +/-257 nm ( Figure 1C) and the nanofibers showed a rough surface with considerable variation in the diameter along the longitudinal axis ( Figure 1D). It can be concluded that the quality of the AN nanofibers is better than RON scaffolds. The rotational collector not only orients the nanofibers but also stretches them to a regular diameter and surface.
The influence of take-up velocity and annealing in the diameter of the nanofibers is depicted in Figure  1E. The RON (zero take-up velocity) presented the highest diameters and standard deviations. The AN 1100m/min and AN 1217m/min without thermal treatment had diameters of 643 +/-50 nm and 747 ±117 nm, respectively. These results were compared with those obtained by Inai et al. [15] with AN of PLA collected at 6 m/min and 630 m/min, these had average diameters of 890+/-190 nm and 610+/-50 nm respectively. It is worth to notice that Inai's experimental electrospinning parameters were different from ours, however, despite of the combination of parameters such as polymer concentration, feed rate, solution electroconductivity, take-up velocity, and type of collectors used in their experiments were different, the AN nanofiber diameters in both works are similar at velocities above 630 m/min. According to our results take-up velocities above 1100 m/min did not influence the nanofiber diameters. In contrast Inai et al. [15], showed that from 6 m/min to 630 m/min (10 fold change in velocity) there is a significant effect in the reduction of the nanofibers diameter. By comparing the annealed and non-annealed nanofiber it was found a small decrement in diameter, ranging between 12 and 30% as shown in Figure 1E. This behavior is due the self-assembly of the chain molecules into the nanofiber as a result of annealing. However this effect was not observed in RON.
The figure 2A shows a phase AFM image of AN prepared without annealing at a take-up velocity of 1217 m/min. Two well-defined alternating (periodic) dark and bright regions appeared on the electrospun nanofiber surface. The dark and bright regions correspond to lamellar and amorphous phases, respectively. Two different phases were clearly identified by using the phase AFM image, captured in tapping mode. This could be due to the difference in the sample hardness, density into the aligned nanofiber. Different regions of the sample were analysed by AFM with highly reproducible results. In the annealed samples, these observed features disappeared. Clearly this observation prove the assumption of Ciceron et al. [17] that represented the morphology of PLA microfibers, produced by melt spinning, as a schematic microfibrillar morphology and confirmed that this model can be used in nanofibers. The lamellar fraction was experimentally measured by AFM and digital image analysis (Image J software), showing an average lamellar volume fraction of 35.7% for AN 1217m/min .

Crystallographic analysis
The XRD plots ( Figure 3) suggest that the level of atomic organization of PLA is strongly dependent on the processing conditions. Bulk PLA, used as a reference, has a semicrystalline structure (purple plot line in Figure 3A) with two characteristic peaks at 16.8° and 19.2° (Bragg angles), in good agreement with those reported by Inai, et al. [15]. These results suggest a crystalline orthorhombic structure (according to PDF-2-2006 card number 00-054-1917, Diffract Plus 2005), with the following lattice parameters a=10.61, b=6.05 and c=28.8 . The randomly oriented nanofibers (RON) have a typical amorphous structure (green plot line in Figure 3A). The fast solidification of the fibers during the electrospinning process limited the long-period three-dimensional order at the atomic level of the PLA; therefore, no evidence of extensive crystallization was found. However, after the fibers were annealed below the melting point (A-RON) a peak at 16.8° appears (red plot line in Figure 3A). This result is a clear evidence of the self-assembly of the polymer chains at the atomic level. Figure 3B shows the XRD results of uniaxially aligned nanofibers (AN). There is no evidence of crystallinity in AN 1100 m/min and AN 1217 m/min as depicted by the blue and black plot lines. However, as it could be expected, annealing promotes extensive crystallization of the polymer chains as shown in the red and green plot lines, where again in both plots the peak is observed at 16.8º. These results are similar to those presented by Inai, et al. [15].
It is most likely that during the electrospinning, the AN polymer chains are oriented along the main axis of the nanofibers due to both the electrical field and winding at high speeds. However, the fast solidification rate tends to inhibit crystallization of the polymer. Annealing promotes chain selfassembly and, as expected, the uniaxially oriented nanofibers (AN) develop more crystallinity than the randomly oriented nanofibers (RON).

Thermal transitions
The thermal transitions of the electrospun nanofibers were studied by DSC in order to measure crystallization trends. The thermal transitions of the electrospun nanofibers are shown in Figure 3C and 3D. It can be observed that the glass transition (Tg) and melting (Tm) temperatures are about the same in all cases: Tg=60 °C and Tm=147 °C. However, the crystallization temperature (Tc) was affected by the take-up velocity. Figure 3C corresponds to bulk PLA, RON and A-RON samples. In this case the RON and A-RON samples, showed an enthalpic relaxation due to the rapid solidification rate during electrospinning. This enthalpic relaxation temperature occurred at about 65°C (endothermic peak). As expected, the degree of molecular disorder of the chains is higher in RON than in A-RON as shown by the enthalpy values of 6.3 J/g and 5.8 J/g, respectively.   [19].
The thermograms of AN and A-AN also show the enthalpic relaxation peaks again at about 65°C, with enthalpy value of 5.8 J/g for both AN 1100 m/min and AN 1217 m/min . This value was similar than that observed in RON. After annealing, the relaxation enthalpies were smaller than in A-RON as follows: 5.8 for A-RON, 4.5 for A-AN 1100 m/min and 3.6 J/g for A-AN 1217m/min . This tendency was also a confirmation that the combination of take-up velocity and annealing parameters improved the internal organization of the polymer nanofibers. The AN also showed crystallization: in the case of AN 1100m/min the crystallization peak was found at 75.3 °C with an enthalpy of 3.9 J/g, while in AN 1217 m/min it was at 74.8 °C with an enthalpy of 4.6 J/g. The orientation of the nanofibers by the effect of the rotational collector induced a significant change in crystallization with respect to RON nanofibers: a reduction of In the case of A-AN the melting enthalpy was greater for the fibers collected at 1217 m/min than those collected at 1100 m/min. In A-AN 1100 m/min and A-AN 1217 m/min the enthalpies were of 18.47 J/g and 29.84 J/g, respectively. These values indicate that a higher take-up velocity improved crystal formation. Our results showed the crystallization peak, and the XRD analysis confirmed the amorphous-crystalline structure in the samples, as it can be observed in Fig. 3. The electrospinning, nanofiber orientation, take-up velocity, and annealing promote self-assembly of the polymer chains.
According to the evidence of XRD ( Figure 3A and 3B), apparently AN nanofibers are not crystalline before annealing. However DSC reveals a crystallization peak in the same type of samples. The XRD technique is not the best to detect the small polymer crystalline structures in the nanofibers in contrast to DSC. In addition to the fact that DSC is a very sensible technique to detect enthalpy changes including crystallization. During the DSC experiments, the heating process induced the oriented polymer chain structures to form bigger crystals before the melting point.

Conclusions
The morphology of scaffolds of electrospun PLA nanofibers depend strongly on the take-up velocity and annealing conditions; winding the electrospun nanofibers in the rotational collector produced highly aligned nanofibers (AN). The morphology of AN was more homogeneous in diameter, without defects, because of nanofiber stretching, than those observed in randomly oriented nanofiber (RON) scaffolds. Fiber stretching produced in turn a high degree of self-assembly. Molecular orientation increases with take-up velocity; however, the fast solidification occurring during electrospinning limited the size of the crystalline structures. Annealing promoted better molecular selfassembly in the nanofibers, increasing the size of the crystals and the degree of crystallization. Aligned nanofiber scaffolds have a hierarchical core-shell structure where the core is amorphous and the shell has a supramolecular morphology as observed by HRTEM. The surface of nanofibers has two periodic phases: the lamellar and amorphous as observed by AFM.
The morphological and structural features found in the nanofiber scaffolds allow to confirm the structures proposed by other researchers with clear and convincing evidence. Also allow to generate a complete description of the structure of the PLA nanofibers and their relationship with variables such as nanofiber orientation on scaffolding, take-up velocity of the collector and subsequent heat treatments to the spun fibers.