8 Additive Manufacturing of 3D Melt-electrospun Star Poly(ε-caprolactone) Scaffolds
Carlos Mota 1, Dario Puppi 1, Matteo Gazzarri 1, Silvia Volpi 1, Cristina Bartoli 1, Dinuccio Dinucci 1, Paulo Bártolo 2, Federica Chiellini 1 and Emo Chiellini 1
1 Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOLab), Department of Chemistry and Industrial Chemistry, University of Pisa, via Vecchia Livornese 1291, 56010 San Piero a Grado (Pi), Italy
2 Centre for Rapid and Sustainable Product Development, Centro Empresarial da Marinha Grande, Rua de Portugal—Zona Industrial, 2430-028 Marinha Grande, Portugal
Abstract
In the last decade, Melt-electrospinning (melt-ES) technique has gained attention for the production of highly porous microfibrous scaffolds for tissue engineering applications. The possibility of processing polymers without the use of organic solvents is one of the main advantages over solution electrospinning. In this study, the processing of linear poly(ε-caprolactone) and star-shaped poly(ε-caprolactone) (*PCL) using a computer-aided melt-ES system equipped with a screw- extruder head is reported. Experimental parameters such as processing temperature, extrusion flow rate and applied voltage were studied and optimised in order to obtain non-woven meshes with uniform fibre morphology. Applying the optimised parameters, the production of three-dimensional scaffolds with a controlled shape was performed with a layer-by-layer approach (0/90º lay-down pattern). *PCL scaffolds composed by five layers were characterized for their cytocompatibility in cell culture experiments using mouse embryo fibroblasts.
Keywords: tissue engineering, scaffold, melt-electrospinning, additive manufacturing, poly(ε-caprolactone), star polymers
8.1 Introduction
The possibility of producing polymeric fibres with a diameter in the microsize range by processing a polymer melt (thus avoiding the use of organic solvents) makes Melt-electrospinning (melt-ES) a technique interesting for tissue engineering (TE) applications [Dalton et al., 2006]. Moreover, the lower packing density of melt-electrospun meshes as compared to nanofibre meshes obtained by solution electrospinning (ES) can allow enhanced cell migration throughout the whole tissue engineered construct [Pham et al., 2006]. However, the achievement of melt-electrospun nanofibres is still a challenge [Góra et al., 2011; Hutmacher and Dalton, 2011]. The production of relatively large fibre diameter and small fibre collection area is an effect of the reduced bending instability of the polymer melt jet in the electric field when compared to solution ES. As largely described in the literature, this phenomenon can be related to the high viscosity and the fast solidification of the polymer melt in the spinning region [Dalton et al., 2007; Karchin et al., 2011; Lyons et al., 2004;
Zhou et al., 2006], and can be favourable to the production of patterned meshes as previously reported by Dalton et al [Dalton et al., 2008]. Direct writing technique indeed envisions the production of melt-electrospun scaffolds with a layer-by-layer approach.
Branched polymers show reduced solution and melt viscosity when compared to linear polymers with equivalent molecular weight [McKee et al., 2005a]. Some studies suggest the application of this type of polymers to produce fibres by ES from solution [Grafahrend et al., 2010; McKee et al., 2005a; Puppi et al., 2010a; Puppi et al., 2010b] or melt [Hunley et al., 2008]. Recent studies performed by our group investigated star-shaped three-arm poly(ε-caprolactone) (*PCL) microstructured constructs fabricated by wet-spinning [Puppi et al., 2011; Puppi et al., 2010b] or solution ES [Puppi et al., 2010b] as scaffolds for bone TE applications. In vitro cell culture experiments using murine preosteoblasts showed good cell adhesion and viability for both kinds of scaffold.
The aim of this work was to apply a novel screw extruder-based melt-ES technique for the fabrication of microstructured scaffolds made of PCL and *PCL with different molecular weight (Mw). The experimental parameters were investigated in order to optimise mesh morphology and fibre diameter. By applying the optimised parameters, *PCL three-dimensional (3D) scaffolds with a controlled geometry and lay-down pattern were manufactured using a layer-by-layer approach.
The developed 3D scaffolds were characterized for their cytocompatibility employing mouse embryo fibroblasts.
8.2 Materials and Methods 8.2.1 Materials
Two batches of three-arm star branched poly(ε-caprolactone) with different average Mw, *PCL64
(Mw = 64000 g/mol) and *PCL189 (Mw = 189000 g/mol), were supplied by Michigan Biotechnology Institute (Lansing, MI, USA). Linear poly(ε-caprolactone) (PCL, CAPA 6500, Mw 50000 g/mol) was obtained from Perstorp Caprolactones Limited (Cheshire, UK).
8.2.2 Melt-flow index (MFI)
Melt-flow index experiments were performed in accordance with ASTM D 1238-04 standard [Materials, 2004] using a capillary rheometer (model 6934, CEAST, Italy). Polymer pellets were loaded in the heating chamber and a piston was placed on top of the polymer to apply a load of 2.16 Kg. All the performed tests were conducted at 80ºC. Before running the test, polymer was pre- loaded without load for 20s and then with load application for 5 s. The polymer was extruded through a die of 2.0955 mm in diameter and 8 mm in length and 15 measures were recorded for each sample.
8.2.3 Melt-electrospinning (Melt-ES)
For the production of scaffolds by melt-ES, a novel melt-extrusion based additive manufacturing (AM) system (Bioextruder), previously described by Domingos et al [Domingos et al., 2009], was used. This apparatus is equipped with a screw extruder to produce 3D structures by processing a polymer melt (Figure 1a).
Figure 1 - Scheme of computer-aided melt-ES: a) screw extruder head, b) translation pattern of the extruder head (0/90º lay down pattern).
For scaffold preparation, polymer was loaded into a reservoir where it was kept at a constant temperature for one hour to allow melting. Afterwards a nitrogen gas pressure (5 bar) was applied to force the polymer melt flowing through the screw extruder that controls the extrusion flow rate (EFR). A 23 gauge (I.D.=340µm) nozzle with a length of 5 mm was used. The extruder is coupled to a DC motor controlling the rotation of the screw. In order to evaluate the correlation between screw rotation speed (SRS) and EFR, each polymer was extruded for one minute at a given SRS, in the range of 7.25 – 29 r.p.m., and the collected polymer fibre was weighted. The experiment was carried out in triplicate at various temperatures in the range 80-160 °C.
A 10x10 cm2 copper plate was positioned on top of the construction platform and used as a collector. By employing a high voltage power supply (SL70P60/230, Spellman High Voltage Electronic Corporation, United Kingdom), the collector was positively charged while the nozzle was electrically grounded. The deposition pattern employed to fabricate layer-by-layer scaffolds was calculated using a Matlab (The MathWorks, Inc.) algorithm. For the present study, a square cuboid model characterized by a base measuring 30 × 30 mm, a distance between the axis of the parallel deposition lines of 2 mm, and 5 overlapped layers (0/90º lay down pattern) was designed (Figure 1b). The nozzle X-Y translational velocity was 500 mm·min−1 and the distance between the nozzle tip and the collector (dNC) was 10 cm. The effect of processing temperature (Tproc) on mesh morphology was investigated in the range 80 to 160ºC, that of applied voltage (Vapp) in the range 15 - 25 kV and that of EFR in the range 0.36 – 6.6 ml·h-1 (by varying the SRS from 7 to 29 r.p.m.).
By employing an auxiliary infrared lamp (Philips, 150W) the ambient temperature and humidity inside the spinning chamber was kept at 35 ± 2 °C and 22 ± 4%, respectively.
8.2.4 Morphological analysis
Mesh morphology was investigated using a scanning electron microscope (SEM; JEOL LSM 5600LV, Japan). The fibre diameter was measured using ImageJ 1.43u software on micrographs with 100x magnification. The average fibre diameter was calculated from over 20 measurements per specimen taken randomly from selected fields.
8.2.5 Biological evaluation
8.2.5.1 Preparation and sterilization of the scaffolds
*PCL189 scaffolds were cut into four pieces of about 1cm2 with an average weight of 0.070 g and sterilized under UV light for 30 minutes each side. Samples were then covered with ethanol
solution (70%) for 48 hours refreshing the solution at 24 hours. Afterwards, samples were extensively washed with phosphate buffer saline (PBS) added with penicillin/streptomycin solution (1%) in order to remove ethanol residues. Samples were pre-incubated for 3 hours with complete Dulbecco’s Modified Eagle Medium before seeding.
8.2.5.2 Cell seeding and culturing
To investigate the ability of the prepared melt-electrospun scaffolds to support cell growth, mouse embryo fibroblast balb/3T3 clone A31 (CCL-163) cell line purchased from American Type Culture Collection (ATCC) was selected. Cells were propagated as indicated by the supplier using α-MEM (Sigma), supplemented with 4 mM of L-glutamine (Sigma), 1% of penicillin:streptomycin solution (10,000 U/ml:10 mg/ml) (Sigma), 10% of calf serum [Sigma] and antimycotic. Cells were maintained at 37°C in a humidified CO2 (5%) atmosphere (Heraeus Instruments).
8.2.5.3 WST-1 tetrazolium salt cell proliferation assay
Cell viability and proliferation were assessed by mean of WST-1 cell proliferation reagent [Roche].
Briefly, the reagent, diluted 1:10 was added to the culture and incubated for 4 hours. Supernatants were then re-plated in 96 well culture plates and analysed with a Biorad microplate reader.
Measurements of formazan absorbance were carried out at 450 nm, with the reference wavelength at 655 nm.
8.2.5.4 Cell viability by direct contact assay
Direct contact assay was performed in order to evaluate the possible leaching of toxic compounds from the investigated scaffolds [Chiellini, 2006]. Briefly, cells were seeded in 24 well plates at a concentration of 2x104 per sample and allowed to proliferate for 48 hours prior to the incubation with the specimens. Then scaffolds were left in direct contact with the near-confluent cell monolayer for other 24 hours and then submitted to the WST-1 assay for the quantitative determination of cell viability. Cells grown onto tissue culture polystyrene (TCPS) were used as control.
8.2.5.5 Cell adhesion and proliferation assay
A preliminary biological evaluation of the suitability of the prepared melt-electrospun scaffolds to sustain cell adhesion and proliferation was carried out as by following: samples were placed in 24
well plate and cells were seeded directly onto the scaffold’s surface at a concentration of 2x104 per sample in a final volume of 0.7 ml, and were then allowed to proliferate for 14 days. After 24 hours from the seeding, samples were transferred in a new plate in order to evaluate the proliferation of only the cells grown onto scaffold. Growth medium was refreshed every 48 hours and the proliferation rate was measured at day 3, 7 and 14, by using the WST-1 assay.
8.2.5.6 Quantification of collagen production
Assessment of collagen production by embryo fibroblasts balb/3T3 clone A31 cultured on *PCL189
scaffolds was carried out at days 3, 7 and 14 and compared with the collagen production of the cells grown in the same culture conditions, but in two-dimensions (2D) on TCPS. At each time points the medium was removed and samples were washed twice with PBS. Scaffolds were then incubated with Direct Red 80 dye (Sigma) dissolved in picric acid (0.1%), for 1 hour at room temperature.
After the incubation time the dye was removed and samples were washed three times with HCl 10 mM to remove dye excess. The elution of the bound stain was performed with NaOH 0.1 N for 30 minutes at 37°C. Supernatants were plated in 96 well plate and the absorbance was read at 540 nm.
*PCL189 scaffold and TCPS with no cells were treated following the same protocol and considered as blank.
The photometric quantification of the collagen was obtained by mean of a calibration curve (Figure 2) prepared with collagen type I, derived from calf’s skin (Sigma). Briefly, known concentrations of collagen in acetic acid (0.1 M) were filmed and dried on glass slides by solvent casting technique, in order to obtain a thin film. Films were then fixed and treated as the samples [Junquiera et al., 1979].
8.2.5.7 Cell morphology investigation by confocal laser scanning microscopy (CLSM)
Morphology of balb/3T3 clone A31 cells grown on the prepared scaffolds and 3D culture organisation were investigated by means of CLSM at days 3, 7 and 14. Cells were fixed with 3.8%
paraformaldehyde for 30 minutes in PBS 1X, permeabilized with a PBS 1X/Triton X-100 solution (0.2%) for 15 minutes and incubated with a solution of 4’-6-diamidino-2-phenylindole (DAPI) (Invitrogen) and phalloidin-AlexaFluor488 (Invitrogen) in PBS for 45 minutes at room temperature in the dark. After dye incubation, samples were extensively washed with PBS 1X and observed by including specimen between two glass coverslips. All steps of the above procedure were performed under gently shaking on an orbital shaker in order to enhance solution penetration into scaffold meshes. A Nikon Eclipse TE2000 inverted microscope equipped with an EZ-C1 confocal laser and Differential Interference Contrast apparatus was used to analyse the samples (Nikon). A 405 nm laser diode (405 nm emission) and an Argon Ion Laser (488 nm emission) were used to excite respectively DAPI and FITC fluorophores. Images were captured with Nikon EZ-C1 software with identical instrumental settings for each sample. Images were further processed with the GIMP (GNU Free Software Foundation) image manipulation software and merged with Nikon ACT-2U software.
8.2.5.8 Morphological observation of cultured cells by scanning electron microscopy (SEM)
Morphological analysis of balb/3T3 clone A31 cells cultured on *PCL189 scaffolds was carried out at day 14. After removal of the culture medium, each cell-cultured scaffolds was rinsed twice with PBS, and the cells were then fixed with 2% glutaraldehyde solution, which was diluted from a 25%
glutaraldehyde solution (Sigma) with PBS 1X, at 1.5 ml/well. After 1 hour of incubation, sample was rinsed again with PBS 1X and then treated with 1.5 ml/well of sodium cacodylate (0.1 M) pH 7.4 for approximately 1 minute. After cell fixation, the specimen was dehydrated in ethanol solution of varying concentration (i.e. 10, 30, 50, 70, 90, and 100%, respectively) for 15 minutes at each concentration. It was then dried in 100% of tetramethylsilane to remove any water traces. The fixed scaffold was mounted on a Scanning Electron Microscopy (SEM) stub, coated with gold, and observed.
8.2.6 Statistical analysis
Quantitative data were presented as mean ± standard deviation (SD). All the in vitro biological tests were performed on triplicate samples. Data sets of processing parameters study and biological characterization were screened by one-way ANOVA and a Tukey test was used for post hoc analysis;
significance was defined at p < 0.05.
8.3 Results and Discussion
8.3.1 Melt-flow index (MFI)
Fibre production using melt-ES is strongly dependent on the polymer rheological properties. MFI value specifies the amount of polymer melt extruded through a die or nozzle during 10 minutes at a specific load and temperature, and is inversely proportional to polymer viscosity. Polymer viscosity and MFI of the different investigated raw materials at 80° C are reported in Table 1.
Table 1 – Melt-flow index (MFI) obtained with capillary rheometer for the different studied polymers at a temperature of 80ºC.
Mw (g/mol) MFI at 80°C (g/10min) Viscosity (Pa·s)
Linear PCL 50000 13.916±0.057 829
*PCL64 64000 239.927±2.526 48
*PCL189 189000 33.857±0.372 341
Results showed that the MFI of linear PCL (Mw=50000 g/mol), when compared with *PCL with lower Mw (64000 g/mol), is significantly lower, while the viscosity is more than 17-fold higher.
Since Dalton et al showed that by decreasing polymer melt viscosity it was possible to reduce melt- electrospun fibre diameter [Dalton et al., 2007], it was possible to hypothesize that *PCL fibres would have shown lower fibre diameter. In order to study the viscosity of the investigated polymers at different Tproc and shear rates, related to the different EFRs employed during melt-ES, further rheological tests should be performed.
8.3.2 Computer-aided melt-ES
Melt-ES of linear PCL and *PCL was investigated by employing an AM system equipped with a screw extruder (Bioextruder).
In comparison to conventional “static” melt-ES, where fibres are collected onto a narrow region, the computer controlled three-axis AM system enables to obtain a more uniform 3D fibres collection and thus to avoid the accumulation of polymeric fibres on a focused area (Figure 3a and 3b).
Figure 3 - Melt-electrospun *PCL189 at a temperature of 160ºC (dNC = 10 cm, Vapp = 25 kV, SRS= 29 r.p.m.):
a) focused deposition by static extruder; b) uniform 3D fibre collection by dynamic extruder (translation velocity = 500 mm·min-1).
8.3.2.1 Evaluation of the polymer extrusion flow rate (EFR)
The used extruder system allows the control of EFR by changing the SRS from 7.25 to 29 r.p.m.
The EFR depends on the processed polymer, the Tproc and the SRS. EFR as function of SRS at three different Tproc is reported in Table 2.
Table 2 - Extrusion flow rate (EFR) of the three investigated polymers at different processing temperatures (Tproc) and screw rotation speed (SRS). Data are expressed as average ± standard deviation (n=3).
For the three polymers investigated, by increasing either the Tproc or the SRS, the EFR increased.
These effects were due to a decrease in viscosity by increasing the temperature and to an increase in shear rate by increasing the SRS. For instance, when linear PCL was processed at 160ºC, by increasing the SRS from 7.25 r.p.m. to 29 r.p.m. the EFR increased from 0.67±0.01 to 2.51±0.06 ml·h-1; when *PCL64 was processed at a SRS of 29 r.p.m,, by increasing the Tproc from 80ºC to 160ºC, the EFR increased from 2.91±0.02 to 6.61±0.07 ml·h-1. However, the effect of Tproc was less pronounced in the case of linear PCL and *PCL189 at higher SRS. In comparison to linear PCL (Mw
= 50000 g/mol), *PCL64 (Mw = 64000 g/mol) showed significantly higher EFR values, while
*PCL189 (Mw = 189000 g/mol) showed comparable EFR values. These results together with the results from MFI characterization support the idea of earlier studies that branched polymers possess
EFR(ml·h-1) SRS
(r.p.m.)
Tproc Tproc Tproc
80ºC 125ºC 160ºC
Linear PCL
7,25 0,36 ±0,02 0,55± 0,00 0,67± 0,01
14,5 1,00± 0,01 1,14± 0,01 1,28± 0,01
21,75 1,66± 0,01 1,76± 0,00 1,90± 0,01
29 2,44± 0,02 2,43±0,02 2,51± 0,06
*PCL64
7,25 0,90±0,01 2,35±0,08 4,16±0,04
14,5 1,59±0,01 2,64±0,07 4,83±0,07
21,75 2,25±0,02 3,27±0,07 5,64±0,07
29 2,91±0,02 3,98±0,03 6,61±0,07
*PCL189
7,25 0,50± 0,03 0,89± 0,01 1,77± 0,01
14,5 1,20± 0,04 1,50± 0,04 2,60± 0,13
21,75 1,80± 0,05 2,19± 0,02 3,18± 0,07
29 2,44± 0,11 2,84± 0,03 3,75± 0,08
a smaller hydrodynamic volume, and consequently lower viscosity, in comparison to linear polymers with equivalent Mw [McKee et al., 2005b; Taskiran et al., 1999].
8.3.2.2 Investigation of processing parameters
Melt-ES of linear poly(ε-caprolactone)
The influence of Vapp (15-25 kV) and EFR (0.36±0.02-2.51±0.06 ml·h−1) over the diameter of melt- electrospun fibres made of linear PCL was investigated at different Tproc (80, 125 and 160°C). The effect of Vapp on extruded PCL filaments was observed only for a Tproc of 160ºC, while for lower Tproc the polymer solidification was too fast to observe such effect (Figure 4).
Figure 4 – Average fibre diameter of melt-electrospun linear PCLat a Tproc of 160ºC and different Vapp and EFR. Error bars represent standard deviation of mean value.
When a Tproc of 160ºC was used, an increase of Vapp from 15 to 25 kV caused a decrease of fibre diameter nearly in the range 37-213µm. By changing SRS, no statistical significant influence was observed comparing data sets obtained for the same Vapp. On other hand, in most of the cases the influence of Vapp, comparing data sets obtained for each SRS tested, showed significant differences in the obtained fibre diameter. A work recently reported by Detta et al [Detta et al., 2010] showed the possibility of producing linear PCL (Mn=80 000 g.mol −1)microfibres by melt-ES employing a syringe pump to control the flow rate. By varying the processing parameters, they developed non- woven meshes with different fibre diameter down to 6 µm (Vapp of 10 kV, dNC of 20 mm, flow rate
of 5 µL·h−1 and a Tproc of 90ºC). In our study it was not possible to produce such small fibre diameter likely because of the larger dNC (10 cm), causing a lower electrical field strength, and/or the much higher flow rate used due to instrument limitations (the lowest EFR was 0.67±0.01 ml·h−1 corresponding to a SRS of7.25 r.p.m. at 160ºC). When the screw was stopped, nitrogen gas pressure was not enough to achieve polymer melt extrusion because of the high viscosity. Further improvements on the reduction of the obtained fibre diameter for linear PCL will be made by reducing the dNC or incrementing Vapp.
Melt-ES of *PCL64
The diameters of *PCL64 melt-electrospun fibres at a Tproc of 80ºC were comparable to those obtained for linear PCL at a temperature of 160ºC (Figure 5). By increasing EFR, the effect of a non-uniform fibre drawing was reduced leading narrower diameter distribution. The differences in diameter of fibres obtained at different EFRs, keeping constant Vapp, were not significant. The effect of Vapp on fibre diameter was significant only comparing samples obtained at 15 and 25 kV and at lower EFRs. The drawing of the fibre induced by the electrical field was not uniform due to the polymer solidification near the nozzle. As consequence fibre diameter was not constant along fibre axis. The process was more stable for a SRS of 29 r.p.m. showing a more uniform fibre diameter, although the standard deviation was still large. An increasing of the Tproc induced the reduction of this problem.
Figure 5 – Fibre diameter of melt-electrospun *PCL obtained at a T of 80ºC and V of 15kV, 20kV and
When *PCL64 was processed at 125ºC, the fibres diameter was in the range 150 µm (Figure 6). By increasing Vapp for a given EFR, a decreasing on the average fibre diameter was observed, although the differences were not significant due to the large diameter distribution. Increase in EFR, keeping constant Vapp, did not induce significant changes on the diameter. A more stable and continuous fibre drawing in the spinning region, resulting in a more uniform fibre diameter, was obtained for higher Vapp and EFR (3.98 ml·h-1 and 25kV, respectively) achieving a fibre diameter of 82±14µm.
Figure 6 – Fibre diameter obtained for *PCL64 with a Tproc of 125ºC and Vapp of 15 kV, 20 kV and 25 kV.
Error bars represent standard deviation of mean value.
When *PCL64 was processed at 160ºC, EFR increased more than two-fold when compared to what achieved at a Tproc of 80ºC (Table 2). The increase of EFR and the decreasing of the polymer viscosity induced a faster travelling of the molten fibre through the air-gap towards the collector.
Therefore when fibres reached the collector they were still in a molten state causing their fusion at the fibre-fibre contact points. Moreover, likely because of the reduced viscosity, the whipping effect was reduced when compared to the lower temperatures tested, causing a bigger concentration of fibres collected in the same area and thus favouring inter-fibre fusion as observed in SEM analysis (Figure 11b). Due to these limitations only the effect of the highest Vapp was tested. Fibres with a diameter (down to 37 µm) and standard deviation significant smaller than those obtained for lower Tproc were achieved (Figure 7). By varying the EFR, significant changes in fibre diameter
were only observed by changing SRS from 7.25 r.p.m. to 21.75 or 29 r.p.m. Further improvements on the fibre collection should be tested in order to avoid inter-fibre fusion. For instance, the increasing of dNC might help to reduce the temperature of the collecting fibre. Other solutions might be the change of the collecting pattern and the deposition velocity used.
Figure 7 – Fibre diameter obtained for *PCL64 at a Tproc of 160ºC and Vapp of 25kV. Error bars represent standard deviation of mean value.
Melt-ES of *PCL189
By increasing the Mw of *PCL, a large increasing of the viscosity was observed (see Table 1). The viscosity of*PCL189 is at least 7 fold higher when compared to the viscosity of *PCL64 at a temperatureof 80ºC (Table 1). This induced an increasing of the diameter of the fibres obtained applying the same processing conditions (Figure 8-10). At a Tproc of 80ºC, fibres diameter was in the range 47-224 µm (Figure 8), depending on the processing conditions, and a large distribution of diameter was observed at lower EFR and Vapp due to the fast solidification of the polymer near the nozzle. However, by increasing Vapp a decrease of the standard deviation was observed. A reduction on the fibre diameter was observed when Vapp was increased at higher EFRs.
Figure 8 – Fibre diameter obtained for * PCL189 with a Tproc of 80ºC, Vapp of 15kV, 20kV and 25kV. Error bars represent standard deviation of the mean value.
In comparison with what observed at 80ºC, the fibres obtained at 125ºC showed smaller fibre diameter in the range 33-172 µm (Figure 9). This small reduction might be correlated to the decreased viscosity and/or to the small increase of the EFR (Table 2) that can diminish the effect of polymer solidification at the nozzle tip. In accordance with what described before, the increase of Vapp and EFR induced a stabilization effect over the fibre collection, and thus a narrowing of diameter distribution.
Figure 9 – Fibre diameter obtained for * PCL189 with a Tproc of 125ºC and Vapp of 15kV, 20kV and 25kV.
Error bars represent standard deviation of mean value.
At a Tproc of 160ºC the process was more stable when compared to lower temperatures and smaller fibre diameters in the range 33-94 µm were obtained (Figure 10). The influence of EFR on the average fibre diameter was not evident. On other hand, the increasing of Vapp originated significant smaller fibre diameters. When the parameters allowing to achieve a stable process at this temperature (7.25 r.p.m.corresponding to 1.77±0.01 ml·h-1, 25 kV) were applied, the fibre presented a diameter of 35±2 µm. A slight variation of the fibre diameter and standard deviation (40±5 µm) was observed when the SRS was increased to 29 r.p.m. (EFR=3.75±0.08 ml·h-1), for a Vapp of 25 kV.
Figure 10 – Fibre diameter obtained for * PCL189 with a Tproc of 160ºC,Vapp of 15kV, 20kV and 25kV. SRS presented on the first line of the X axis of the graph and correspondent EFR in the second line. Error bars represent standard deviation of mean value.
Overall, the undertaken research showed that the morphology of fibres and fibrous collections fabricated by melt-ES is influenced by different parameters, such as dNC, electrical field, flow rate, polymer viscosity and Tproc. In this study the feasibility of producing melt-electrospun fibres of linear PCL and *PCL with two different molecular weights was investigated.
8.3.2.3 Effect of the applied voltage (V
app)
Generally an increasing in Vapp induced a reduction of size and distribution of fibre diameter because of the higher and more uniform stretching force acting on the ES jet. For instance, in the case of linear PCL processed at the maximum SRS (29 r.p.m, EFR=2.51±0.06 ml·h-1, Tproc=160ºC)
it was possible to produce fibres with a diameters of 182±11, 148±11 and 131±14 µm employing a Vapp of 15, 20 and 25 kV, respectively. The same trend was observed for *PCL polymers. As an example, *PCL189 fibres diameter for a Tproc of 160ºC was significantly affected by Vapp: the diameter obtained with an SRS of 29 r.p.m. (EFR= 3.75±0.08 ml·h-1) decreased with values of 70±9, 54±6 and 40±5 µm for Vapp of 15, 20 and 25 kV, respectively.
8.3.2.4 Effect of processing temperature (T
proc)
Tproc was the most influent parameter for the three investigated polymers. With the increasing of Tproc a significant reduction of the polymer viscosity, and thus of the rheological behaviour of the polymer during the processing, is induced. Moreover, the temperature profile in the needle- collector spinning gap is also reported to greatly affect the solidification behaviour of the polymer jet [Zhou et al., 2006]. In our study the effect of the Tproc in the screw extruder head was investigated in the range 80ºC to 160ºC, and the temperature in the spinning zone was kept constant at 35 ± 2 °C. The effect of the electrical field on melt filament of linear PCL was observed only at a Tproc of 160ºC. For this temperature fibre size varied from 197 ±15 µm (Vapp = 15 kV, 7.25 r.p.m.) to 131±14 µm (Vapp = 25 kV, 29 r.p.m.).
The optimal Tproc for *PCL64 was 125ºC due to the fusion of the collected fibre observed for higher temperatures. In this case the fibre diameter varied from 108 ±47µm (Vapp=15 kV, SRS=7.25 r.p.m.
corresponding to EFR = 2.35±0.08 ml·h-1) to 81±13 µm (Vapp = 25 kV, SRS = 29 r.p.m.
corresponding to EFR = 3.98±0.03 ml·h-1). Smaller diameters were obtained at Tproc of 160ºC although fibre-fibre fusion occurred. The thinnest fibres obtained for this temperature had a diameter of 43±7µm. In the case of *PCL189, a Tproc of 160 °C allowed to obtain a uniform fibrous structure with a fibre diameter from 65±13 µm (Vapp = 15 kV, 7.25 r.p.m. corresponding to 1.77±0.01 ml·h-1) to 40±5 µm (Vapp = 25 kV, 29 r.p.m. corresponding to 3.75±0.08 ml·h-1).
The high residence periods of the polymer at high temperature might induce in some cases thermal degradation or polymer depolymerisation. The extrusion head used is composed of two melting zones controlled by two proportional–integral–derivative (PID) controllers that allow an accurate and independent control of the temperature in the reservoir and the screw-extruder. This allows a pre-heating of the polymer over its melting temperature and then an increasing of temperature in the screw extruder body, thus minimizing the period of time that the polymer is subjected to high processing temperatures.
8.3.2.5 Effect of extrusion flow rate (EFR)
The EFR of the screw extruder used in this study was evaluated for different Tproc and SRS.
However, the influence of the EFR on melt-electrospun meshes morphology was not clear in most of the cases. When a uniform stretching of the polymer jet in the spinning region was not achieved, the large standard deviation of the fibre size obtained hampered a correct analysis over the effect of the EFR (controlled by the SRS). In most of the cases, when the process was more stable no significant diameter changes were observed by the EFR in the range tested.
In our studies, the use of a screw extruder system allowed to produce melt-electrospun fibres at high EFR. Although, these results differ from those published by Lyons et al [Lyons et al., 2004]
who observed that the use of a lab scale Brabender extruder limited the production of melt- electrospun fibres due to the high EFR of the polymer. In our experiments, when the extruder was stopped, in most of the cases the gas pressure was not sufficient to force the polymer melt flowing and no melt-electrospun fibre was thus generated. This was likely due to the high polymer viscosity and/or the low electrical field strength applied. An exception was observed for *PCL64 at a Tproc of 160ºC. However, even if in this case melt-electrospun meshes were produced at lower EFR, they presented fusion at the fibre-fibre contact points like in the case of screw rotation-driven extrusion at the same Tproc.
8.3.2.6 Effect of other processing parameters
The control over the ambient conditions (i.e. temperature and humidity) is fundamental in solution ES and melt-ES experiments, even if it is ignored in most of studies. The increment of the ambient temperature during melt-ES delays the solidification of the fibre when the elongation is taking place in spinning region, increasing the fusion at the fibre-fibre contact points and reducing the obtained fibre diameter. A study of the influence of the ambient temperature was not performed due to lack of an accurate control over the infrared lamp intensity used in our system and further studies with an improved control should be carried.
The dNC is also a parameter that generally influences the obtained fibre diameter. Since our goal was to produce scaffolds composed of melt-electrospun aligned fibres, that was not possible in this study due to the low motion speed of the extruder head, improvements of the equipment to achieve a faster translation of the head is necessary. This will allow the obtainment of straight fibres avoiding the collection in a coiled form (see Figure 13).
8.3.2.7 Morphological analysis
Representative micrographs of melt-electrospun meshes made of the three investigated polymers, obtained by applying the optimised processing conditions, are shown in Figure 11. The developed scaffolds were characterized by a 3D non-woven structure with pore size in the range of tens to hundreds of micrometres and limited fusion at the fibre-fibre contact points.
The diameter of the fibres produced in this study was on the micro size range. In comparison to the typical nanofibrous network by solution ES, the lower surface area/volume ratio of the obtained meshes can result in lower cell attachment. However, the lower packing density can be favourable to an enhanced migration of the cells throughout the whole scaffold. In addition, the larger pores size can allow a better exchange of nutrients and metabolic by-products as well as higher migration of cells from the host surrounding tissues and neovascularisation in vivo. Pham et al [Pham et al., 2006] showed that an increase of fibre diameter from nano- to micro-size, improved cell migration throughout the scaffold.
Figure 11 – SEM micrograph of melt-electrospun scaffolds produced with Vapp of 25 kV and SRS of 29 r.p.m.: a) linear PCL produced with Tproc of 160ºC, b) * PCL64 produced with Tproc of 125ºC,and c) * PCL189
produced with Tproc of 160ºC. Inset images with detail of fibre surface topography obtained, scale bar of 10 µm.
8.3.2.8 Layer-by-layer fabrication of 3D melt-electrospun scaffolds
By applying the processing conditions optimised for the three kinds of polymers, 3D scaffolds were fabricated layer-by-layer by depositing the melt-electrospun fibre with a predefined 0-90° lay-down pattern (Figure 12).
Figure 12 - *PCL189 melt-electrospun scaffolds composed of five layers sliced into four pieces (1cm2) for cell culture experiments.
As largely described in literature [Dalton et al., 2007; Karchin et al., 2011; Lyons et al., 2004], the limited bending instability of the jet, related to the high viscosity of the polymer melt, limits the production of a uniform 3D fibre mesh by melt-ES. However, a low bending instability could be seen as an advantage for the controlled deposition of a melt fibre, as reported by Dalton et al [Dalton et al., 2008] in a work employing a manually-translated collector for the manufacturing of patterned melt-electrospun substrates. In our approach, a computer-controlled system that allowed the collection of the fibres with a predefined 0/90º lay-down pattern was employed achieving enhanced accuracy and reproducibility of mesh morphology. This allowed the manufacturing of 3D scaffolds with a layer-by-layer principle. The process stability, i.e. the continuous and uniform drawing of the melt polymer jet in the spinning region, is influenced by different processing parameters as previously reported. During the fabrication by AM of PCL and *PCL scaffolds, the whipping phenomenon was only observed near the collector and the fibre was collected in a coiled form by following the pattern of deposition (Figure 13).
Figure 13 – Melt-electrospun fibres collected in a coiled form following the deposition pattern. Processing conditions:*PCL189 with a Tproc of 160ºC, SRS of 29 r.p.m. and Vapp of 25 kV. Arrow indicates the deposition direction.
3D *PCL189 scaffolds fabricated by employing the optimised parameters (T = 160ºC, EFR=3.7±0.08 ml·h-1, Vapp = 25 kV) composed of five layers (Figure 1b, 12) were characterized for their compatibility with a fibroblast cell line.
8.3.3 Biological characterization
Most TE strategies for replacement of functional tissues or organs rely on the application of 3D scaffolds that guide the proliferation and spreading of seeded cells in vitro and in vivo. In fabricating scaffolds for TE, the nano and microscaled architectures should be considered, in the view of biological applications.. A biologically inspired scaffold fabrication approach for wound healing is to create extracellular matrix (ECM) analogues composed of nanoscale fibres with mimicking structure and functions to native ECM. The ECM influences cell differentiation, proliferation, survival, and migration through both biochemical interactions (cell adhesion, presentation of growth factors) and mechanical cues (stiffness, deformability) [Gieni and Hendzel, 2008; Owen and Shoichet, 2010]. The melt-ES process has been proposed as a highly promising technology producing small diameter fibres as narrow as several hundreds of nanometres, which mimic the nanofibrous structure of the natural ECM, by eliminating potentially cytotoxic solvents derivable from the manufacturing process [Hutmacher and Dalton, 2011; Karchin et al., 2011; Park et al., 2008]. Electrospun scaffolds provide high surface area to volume ratio, which has been
proved to promote cell-matrix interaction at the nanoscale. Moreover, this characteristic facilitates oxygen permeability and allows fluid accumulation, which is highly desirable in wound healing process [Zhong et al., 2010].
8.3.3.1 Cell viability and proliferation
The direct contact assay performed on *PCL189 scaffolds showed the absence of toxic compounds released from the scaffolds. The obtained results displayed values of cell viability comparable to the control, as shown in Figure 14.
Figure 14 – Cell viability of balb/3T3 clone A31 cultured in direct contact with *PCL189 based scaffolds, evaluated by WST-1 assay.
Quantitative evaluation of proliferation of cells seeded onto the prepared scaffolds, performed by WST-1 assay at day 3, 7 and 14, highlighted a significant increase of cell proliferation during the culturing period (Figure 15), assuming a complete colonisation of the scaffolds.
Figure 15 – Cell proliferation of balb/3T3 clone A31 cultured onto *PCL189 scaffolds, evaluated by WST-1 assay.
Overall, preliminary biological evaluations suggested the suitability of these scaffolds to sustain the balb/3T3 clone A31 adhesion and proliferation, with a promising role as biomimetic 3D ECM providing structural support to cells and a milieu for cell migration [Owen and Shoichet, 2010].
8.3.3.2 Collagen production
A simple and reproducible method to estimate collagen synthesis involves the use of Direct Red 80 dye. This dye has been used since 1964 for staining collagen in histologic specimens [Lopez-De Leon and Rojkind, 1985; Taskiran et al., 1999], and has been applied in measuring soluble collagen that had been fixed to glass slide [Junquiera et al., 1979]. Moreover, Lee et al. reported the use of the dye as a system to bind and quantify triple helical collagen molecules produced from fibroblast- like cells [Lee et al., 1998]. It is known from the literature [Taskiran et al., 1999] that direct red is specific for many collagen’s types (I, III, IV and V) and gives similar calibration curves for each collagen type. In the present study, type I collagen was selected for the calibration of the assay because of the predominance of the type I (90%) in connective tissues.
According to this method, collagen content of balb/3T3 clone A31 cells, cultured on melt- electrospun *PCL189 scaffolds and on TCPS, was measured and quantified, as reported in the Figure 16. Values of collagen expressed as micrograms and obtained from A31 cultured on 3D
*PCL189 constructs and 2D supports have been compared on the basis of the same values of cell proliferation expressed as absorbance of produced formazan measured at 450 nm. After the first three days of culture the amount of collagen was comparable between 2D and 3D conditions.
Nevertheless, for longer culture times A31 cells cultured on *PCL189 meshes showed an increased production of collagen, 4-8 time higher than cells cultured on TCPS.
Figure 16 - Collagen production from balb/3T3 clone A31 cultured onto *PCL189 meshes and TCPS.
Thus, *PCL189 scaffolds displayed a high capability to induce A31 cell to promote the production of collagen proteins, confirming the biocompatibility and the suitability of the prepared scaffolds as ECM substitutes.
8.3.3.3 Cell morphology investigation by confocal laser scanning microscopy (CLSM)
A preliminary biological evaluation of the prepared *PCL189 scaffolds to sustain cell adhesion and proliferation was carried out also by a qualitative point of view. Fluorescent staining of cytoskeleton and nuclei showed morphology of A31 cells and colonization of the melt-electrospun scaffolds during the culturing period (Figure 17).
Figure 17 - CLSM images (magnification X10 and X20) of balb/3T3 clone A31 cultured on *PCL189 scaffolds
At the third day of culture, microscopic observation showed a reduced presence of cell clusters adherent on fibres’ surface in agreement with measured viability. This behaviour could be due to the inefficacy of the seeding procedure, because of the inability of the polymeric meshes to retain a large number of cells. Nevertheless the analysis for longer culture time points (7 and 14 days) showed a progressive increase of cells colonizing polymeric meshes, as previously demonstrated from cell proliferation assay. At day 14 cultured samples exhibited full cellular colonization of available fibres’ surface by a wide continuous cell culture net. The cells, observed with a 20X magnification, displayed a spread out, both fusiform and stellate shapes with dendritic (arm-like) extensions from the cell membrane anchoring the cells to the scaffold’s surface. Cell architecture showed consistent F-actin organisation with early stages of cell adaptation to the material [Hutmacher et al., 2001], exhibiting great stress fibres stretched along the cytoplasm.
8.3.3.4 Morphological observation of cell-cultured scaffolds by SEM analysis
SEM analysis allowed for the further characterization of the morphology and colonization of the balb/3T3 clone A31 cultured for 14 days on the *PCL189 scaffolds. As shown in Figure 18, A31 cells showed features indicative of cell activation, including numerous filopodia and fibre-like processes that allowed the anchorage of the cells to the substrate with the formation of a complex multicellular coverage [Zhang et al., 2011].
Figure 18 - SEM images of balb/3T3 clone A31grown on melt-electrospun scaffolds. a) scale bar of 100 µm, b) scale bar of 50 µm, c) and d) scale bar of 20 µm.
A31 cells seemed to well colonize the roughness of the fibre surface by highlighting an evident adhesion and spreading, confirming the previous data of cell proliferation and CLSM.
8.4 Conclusions
In the present study, 3D melt-electrospun meshes were successfully produced from linear PCL,
*PCL64 and *PCL189. Star-shaped polymers were efficiently melt-electrospun at lower Tproc in comparison to linear PCL. The Tproc was the most influent factor on scaffold morphology and the obtained fibre diameter. In spite of the relatively large fibre diameter obtained, it was possible to produce fibres with a diameter smaller than 40 µm. Scaffolds were produced in an additive way (layer-by-layer) with five layers. A good control over fibre collection was achieved, although further improvements should be performed in order to collect straight fibres in an accurate and controlled way.
A preliminary screening was carried out in order to evaluate the suitability of the scaffolds as 3D supports in the replacement of the natural ECM. Mouse embryo fibroblast balb/3T3 clone A31 has been selected as cellular model. These scaffolds have demonstrated to be able to support cell adhesion, proliferation and migration, preserving and increasing the ability of the cells to produce collagen, one of the main fibrillar component of the ECM [Dutta and Dutta, 2010]. Future studies will be devoted to better investigate the suitability of the melt-electrospun *PCL scaffolds as ECM substitutes for wound dressing applications [Zhong et al., 2010]. In this respect, co-cultures of dermal fibroblasts and keratinocytes will be used in order to verify the suitability of these constructs to provide structural integrity and mechanical strength to skin tissues in the view of the wound healing process [Gümüşderelioğlu et al., 2011].
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