D. M. Correiaab,
R. Gonçalvesab,
C. Ribeiroac,
V. Sencadasad,
G. Botelhob,
J. L. Gomez Ribellesef and
S. Lanceros-Méndez*ac
aCentro/Departamento de Física da Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. E-mail: lanceros@fisica.uminho.pt
bCentro/Departamento de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
cINL – International Iberian Nanotechnology Laboratory, 4715-330 Braga, Portugal
dEscola Superior de Tecnologia, Instituto Politécnico do Cávado e do Ave, Campus do IPCA, 4750-810, Barcelos, Portugal
eCenter for Biomaterials and Tissue Engineering, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain
fNetworking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Valencia, Spain
First published on 17th July 2014
Poly(vinylidene fluoride) (PVDF) microparticles have been produced by electrospraying as a suitable substrate for tissue engineering applications. The influence of the polymer solution concentration and processing parameters, such as electric field, flow rate and inner needle diameter, on microparticle size and distribution has been studied. Polymer concentration is the most influential parameter on PVDF microparticle formation. Higher concentrations promote the formation of fibers while dilute or semi dilute concentrations favor the formation of PVDF microparticles with average diameters ranging between 0.81 ± 0.34 and 5.55 ± 2.34 μm. Once the formation of microparticles is achieved, no significant differences were found with the variation of other electrospray processing parameters. The electroactive β-phase content, between 63 and 74%, and the crystalline phase content, between 45 and 55%, are mainly independent of the processing parameters. Finally, MC-3T3-E1 cell adhesion on the PVDF microparticles is assessed, indicating their potential use for biomedical applications.
Polymer microparticles have found applicability in biomedical engineering for drug delivery systems5,6 and are increasingly being used as supports for cell expansion and differentiation, which implies the control over micro and macrostructural features of the polymer substrate;7 scaffolds formed by polymer microparticulates can hold and populate more cells than the traditional 3D scaffolds.7
It has been shown that electroactive polymers, in particular piezoelectric poly(vinylidene fluoride) (PVDF), that generate an electrical signal in response to mechanical loads, can be used as a bioactive electrically responsive material as a promising approach for improving tissue engineering strategies,8–10 as electrical stimulation influences cell proliferation, differentiation and regeneration.11,12
PVDF is a semi-crystalline polymer that is receiving increasing attention as a support for cell culture due to its strong piezoelectric properties, high mechanical strength, thermal stability, chemical resistance and high hydrophobicity properties.11,13–15 This polymer has at least four crystalline structures (α, β, γ and δ), being the β-phase the one with the largest piezoelectric response, which allows applications in the areas of sensors and actuators, energy generation and storage and, due to its biocompatibility, also in biomedical applications and tissue engineering.11,13–15
Electrospray is a promising technique for preparation of polymeric micro- and nanoparticles.16 This method might overcome some of the drawbacks associated with conventional microparticle-producing methods such as solvent casting, single and double emulsion, spray-drying, porous glass membrane emulsification and coacervation.16
The principles of electrospray are similar to the ones of the electrospinning process. In electrospray, polymer microparticles can be produced from a polymer solution in a conductive enough solvent. The variation of solution properties such as concentration, viscosity and surface tension, and processing parameters, such as flow rate, needle diameter, distance of the needle to the collector and applied voltage, promotes the formation of a continuous jet that can be broken down into droplets, resulting in microparticles of different size.16 The advantage of electrospray is the fact that the droplet size can be controlled by adjusting solution and processing parameters.17
Natural and synthetic polymers have been processed in the form of microparticles by electrospray.18 The most common natural polymers produced by electrospray are gelatin,19 chitosan18,20 and elastin.18 Gelatin19 and chitosan20 microparticle aggregates have been used as a 3D scaffold in cartilage tissue engineering. Synthetic polymers including polylactides (PLAs), poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) have also been electrosprayed.18 Among other studies, PLGA microparticles of 4–5 μm in average diameter have been used as a drug delivery system for bone tissue regeneration.21
Despite to the interest of using electroactive microparticles for several applications, to our knowledge, there is just one report on the use of electrospray to prepare thin PVDF films composed by PVDF microparticles with diameters in the range of 61 to 250 nm.17
Thus, this work reports on the production of PVDF microparticles by electrospray. By controlling solution parameters, namely polymer concentration, a stable process has been achieved allowing to obtaining microparticles with controlled size. The suitability of the developed microparticles as a substrate for tissue engineering application was proven by cell viability studies performed with osteoblast-like MC3T3-E1 cells.
The polymer was dissolved in a DMF–THF co-solvent system with a volume ratio of 85/15 (v/v) for PVDF concentrations of 5, 7 and 10 (% w/v). THF is selected by its lower boiling point, when compared to the DMF solvent. The ratio DMF–THF was chosen after a series of experimental measurements taken into account the polymer microspheres integrity and jet stability. The solutions were kept under agitation with a magnetic stirrer at room temperature until complete dissolution of the polymer.
For cell culture, 10 mg of microparticles obtained by electrospray (7% w/v) were placed in a 2 mL Eppendorf. For sterilization purposes, the microparticles were immersed in 70% ethanol, followed by washing with phosphate-buffered saline solution (PBS) 5 times for 10 min under constant shaking. Before cell seeding, fibronectin (FN) was adsorbed by immersing the microparticles in a FN solution of 20 μg mL−1 overnight under constant shaking.
For the cell viability study, MC3T3-E1 cells (density of 1.5 × 105 cells per Eppendorf) were mixed with the microparticles up to 3 days. Cell pellets without any microparticles were used as reference (control +) and only microparticles were used as negative control. For the quantification of cell viability, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT, Sigma-Aldrich) assay was carried out. MTT is used to measure the number of metabolic active cells based on the quantification of the activity of living cells via mitochondrial dehydrogenases. At each time point, the supernatant was removed and fresh medium containing MTT solution was added to each Eppendorf. After 3 h of incubation, the supernatant was removed and dimethyl sulfoxide (DMSO) was added to dissolve the MTT formazan crystals. Thereafter, the solution of each Eppendorf was mixed in a shaker in order to exclude the microparticles and the supernatant was used to determine the absorbance at 570 nm. Three measurements were performed for each sample.
Fig. 1 shows representative SEM images of the PVDF microparticles prepared by electrospray from a 2, 5, 7 and 10 (% w/v) solution concentration using a solvent mixture of THF and DMF. The corresponding microparticle size distribution is also shown in Fig. 1. Electrospray from low polymer concentration solution (2% w/v) did not result in microparticles with spherical geometry (Fig. 1a), a fact that can be ascribed to the low polymer content in the jet leading to low solution viscosity and high surface tensions of the solution. Under those conditions no polymer entanglement is achieved.22 Further, diluted polymer concentrations solutions favors the formation of tailed microparticles (Fig. 1a) due to the lack of sufficiently strong polymer chain entanglements.22 On the other hand, spherical PVDF microparticles with different size distributions were obtained with polymer concentrations 5 (% w/v) or more, as reported in ref. 23.
The spherical morphology of the PVDF microparticles is attributed to the complete solvent evaporation from the droplets before reaching the collector, complementary to the polymer diffusion during solvent evaporation. A rapid polymer diffusion ensures the achievement of solid and dense microparticles but does not necessarily lead to the spherical morphology.6,18
PVDF microparticles obtained from the dissolution of PVDF in the co-solvents of DMF–THF are compact due the low boiling point of THF that allows fast polymer crystallization from the liquid jet surface. The high boiling point and low vapor pressure of DMF hinders fast solvent evaporation and promotes a decrease of the mean microparticle size, leading to dense polymer microparticles (Fig. 1).18 Moreover, moisture present in the atmosphere when the electrospray is carried on, also contributes to the high surface roughness observed in Fig. 1. It has been shown in different polymer systems that high moisture levels present during electrospinning favors the presence of circular pores on top of the electrospun fibers, that become larger with increasing humidity until the coalescence to form large, non-uniform shaped structures.24,25
At a concentration of 7 (% w/v) and more some thin fibers were detected among polymer microparticles and it was observed when the polymer concentration increases, the amount of fibers in the collector increases and, consequently, the amount of polymer microparticles decreases. This is related to the facts observed in26 in which smooth and beadles PVDF fibers were obtained for polymer concentrations above 20 (% w/v), leading to some beads in the fiber mats for lower polymer concentrations. Fiber formation has been also reported for polymer concentrations above 10 (% w/v),23 attributed to enough solution viscosity and surface tension that favors polymer chain entanglement. Our results suggest that dilute or semi-dilutes solutions favor the formation of polymer microparticles while fibers are formed for concentrated solutions above 10 (% w/v) as reported in ref. 27.
In this sense, polymer concentration plays a central role in fiber or microparticle formation and therefore in process optimization.18 Thus, the ideal regime of polymer solution to obtain microparticles is the semi-dilute moderately entangled, where a significant degree of entanglement is observed and dense, solid and reproducible microparticles are obtained.18 In this state, it is essential that the concentration of the solution (c) is larger than the critical entanglement concentration (cent) but lower than the critical chain overlap concentration (cov): for c > 3cov the regime is defined as semi-dilute highly entangled regime and characterized by the presence of beaded fibers or fibers. In this sense, for a PVDF concentration of 10 (% w/v), the solution is in a semi-dilute highly entangled regime,6,18 being the critical entanglement concentration around 5 (% w/v), in order to achieve PVDF spherical microparticles. The critical chain overlap is observed when the droplet carries enough polymer to overlap, but not sufficient to generate a significant degree of entanglement, giving origin to deformed particles and non-uniform and non-reproducible morphology. According to our results, cov is around 2 (% w/v). The influence of polymer concentration (5, 7 and 10 (% w/v)) in the average size of the PVDF microparticles is shown in Fig. 2, resulting in an increase in the average microparticle diameter from 2.5 to 5.5 μm, with the increasing polymer concentration.
The average size of microparticles for the different needle inner diameters was evaluated (Fig. 4c) and the results show a quite similar microparticle diameter for all the different samples with an average size diameter between 2.0 and 2.5 μm, being therefore independent of the needle inner diameter.
Fig. 5 summarizes the influence of the polymer concentration on the size of electrosprayed polymer microparticles, as this is the parameter that mainly influences microparticle formation and diameter (Fig. 1). Morphology and size of microparticles can be further tuned by the additional parameters described above in order to obtain fibers without beads and in the case of microparticles the absence of fibers.
The chemical structure of PVDF is composed by the repetition unit–CH2–CF2– along the polymer chain and characteristic vibrational modes can be used to the identification of the α and β phases.15,29 The α-phase can be identified by the presence of absorption bands at 489, 530, 615 and 766 cm−1 attributed to stretching of the group CF2, at 795 cm−1 corresponding to the CH2 stretching and at 855 and 976 cm−1 resulting of the CH group stretching.30 The β-phase content present in the sample can be determined by the absorption infrared band at 840 cm−1 corresponding to the stretching of the CH2 absorption band and by 511 and 600 cm−1 characteristic of CF2 and CF stretching, respectively.15,30
Fig. 6a shows FTIR-ATR spectrum of representative PVDF electrospray microparticles and α-PVDF film for comparison. Electroactive β-phase is desired for sensor and actuator applications as well as for tissue and biomedical engineering due to its piezoelectric properties that enhances cell growth and proliferation.12,14 Fig. 6a shows that the characteristic bands of β-PVDF and α-phase are present in the polymer microparticles. This fact has been previously reported for electrospun PVDF fibers and their composites15,26 and was attributed to the combination of low solvent evaporation and electric field stretching of the fibers.30 It the case of microparticles produced by electrospray the presence of the electroactive phase is due to the low temperature solvent evaporation.
The evolution of the β-phase content of the microparticles was determined by the eqn (1), as explained elsewere:15,29
![]() | (1) |
Fig. 6b and c shows that electrospray processing parameters does not influence substantially the amount of β-phase content present in the sample as phase content is mainly determined by the low crystallization temperature (room temperature) that favors polymer crystallization in the electroactive phase.28
![]() | (2) |
The viability of MC3T3-E1 cells seeded in an Eppendorf with and without PVDF microparticles was examined by MTT assay (Fig. 9). The obtained results reveal that the cell agglomerates are viable for both. Comparing the PVDF microparticles/cells pellet with the cell pellet used as control it is possible to verify a higher number of cells after 72 h on the pellet with microparticles. This result shows that the PVDF microparticles can provide a suitable environment for cell growth, than can be further explored through suitable mechanical stimulation leading to electromechanical response of the microparticles.14
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Fig. 9 Cell viability for PVDF microparticles/cells and cells pellets (control +). Results are expressed as mean ± standard deviation with n = 3. |
Infrared spectroscopy showed that electrospray allows the processing of the PVDF microparticles in the β-phase, with electroactive phase contents of around 70%. Moreover, processing parameters does not influence substantially the amount of β-phase content.
DSC results of the PVDF microparticles show that the co-solvents used during the PVDF dissolution and the varying electrospray processing conditions allow variations in the degree of crystallinity between 47 and 57%, being the melting temperature of the samples independent on the processing conditions.
MC-3T3-E1 cell adhesion was not inhibited by the PVDF microparticles preparation, indicating the suitability of the material for the development of electroactive scaffold for biomedical applications.
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