Piezoelectric PU/PVDF Electrospun Scaffolds For Wound Healing Applications

Background and objectivesWound healing is a process in which the skin (or other tissues) repairs itself afterinjury. The process can be accelerated and enhanced by the use of wound dressings. Thusfar, different types of wound dressings have been developed based on the specific material,structure and drug that are favourable for wound healing. However, wound dressings basedon electrical stimulation have not been reported yet.Electrical stimulation influences cell behaviors such as proliferation, differentiationand regeneration. A number of previous studies have shown that piezoelectric materials thatgenerate electrical charges in response to mechanical strain may be used to preparebioactive electrically charged surfaces. Polyvinylidene fluoride (PVDF) is a piezoelectricpolymer that exists in at least three regular phases: α, β and γ phases, etc. Common freeradical polymerisation-processed PVDF is typically in the nonpiezoelectric α phase. Toobtain the piezoelectric β phase, the α phase PVDF needs to be mechanically stretched toorient the molecular chains and then poled under tension. Reports have indicated thatelectrospinning is a simple technique to form the the piezoelectric β phase of PVDF directlyfrom solution.Electrospinning is an attractive approach for the fabrication of fibers with diametersranging from a few nanometers to several micrometers by creating an electrically chargedjet of polymer solution or melt. This technique has been recently introduced as the mostpromising technique to manufacture scaffolds for tissue engineering applications. Thescaffolds could partially mimic the structure and function of natural extracellular matrices(ECM), thereby enhancing cell adhesion via1) the interconnectivity of voids favourable forcell in-growth and2) the high surface area to volume ratio, which enlarges cell-scaffoldinterface.A recent study has reported the preparation and in vitro cytocompatibility of poly (vinylidene fluoride-trifluoroethylene)(PVDF-TrFE) electrospun scaffolds and argued thatthere is tremendous potential of the scaffolds for tissue engineering applications. However,the exact piezoelectric effect on the cultured cells was not investigated. In the current study,PU/PVDF scaffolds are prepared by electrospinning. Polyurethane (PU) is a thermoplasticelastomer that has been widely used as prostheses. It is co-electrospun with PVDF becauseof the improved elasticity of the resulting scaffolds. The crystalline phase of PVDF in thescaffolds is characterized, and the piezoelectric effect on fibroblast activities in vitro and invivo are thoroughly investigated with the aim of demonstrating the possible application forwound healing.Methods1. PU/PVDF scaffolds of different composition ratios ranging from1:3to3:1(V/V)were prepared by electrospinning.2. The tensile strength and elongation at break of the scaffolds were tested using anelectronic universal testing machine. The piezoelectric coefficient (d33) of the scaffoldswere tested using Thin/Thick film Piezoelectric Analyzer. Scaffolds that kept a balancebetween mechanical property and piezoelectric property were used for the followingexperiments. The crystalline phase of PVDF in the scaffolds was characterized by X-raydiffraction (XRD), differential scanning calorimetry (DSC) and Fourier transform infraredspectroscopy (FTIR), respectively.3. NIH3T3cells were cultured on the scaffolds. Cell morphologies on the PU/PVDFscaffolds were observed by laser scanning confocal microscopy (LSCM) and scanningelectron microscopy (SEM). CCK-8assays were performed to compare thecytocompatibility of the scaffolds with that of the tissue culture polystyrene (TCPS).4. To excite piezoelectricity, BioFlex Culture Plates were used with FX-4000TFlexercell tension plus system. The scaffolds were stuck to the flexible silicone bottom ofthe culture plates. Pressure applied to the silicone membrane was then transmitted to thescaffolds, thereby exciting their piezoelectricity. Wound-healing assay, cell-adhesion assay,quantitative RT-PCR and Western blot analyses were performed to investigate piezoelectriceffect of the scaffolds on fibroblast activities.5. The scaffolds were subcutaneously implanted in Sprague-Dawley (SD) rats to verifytheir biocompatibility. SEM, HE staining, LSCM, flow cytometry (FCM) and mechanical testing were performed to investigate the piezoelectric effect of the scaffolds on fibrosis invivo.Results1. The parameters for bead-free scaffolds produced from the PU/PVDF solutions wereas follows: flow rate0.8ml/h, applied voltage15kV and collecting distance20cm. ThePU/PVDF (1:1) scaffolds were used for the following experiments. The mean fiberdiameter and pore size of the scaffolds were1.41±0.32μm and11.47±1.14μm, respectively.XRD, DSC and FTIR analyses had revealed that the electrospinning process had altered thePVDF crystalline phase from the α phase to the β phase.2. SEM and LSCM revealed that NIH3T3cells grew and proliferated normally on thePU/PVDF scaffolds. CCK-8assays revealed that the cytocompatibility of the scaffolds wascomparable to that of the TCPS.3. Wound-healing assay, cell-adhesion assay, quantitative RT-PCR and Western blotanalyses revealed that the fibroblasts cultured on the piezoelectric-excited PU/PVDFscaffolds showed enhanced migration, adhesion and secretion. The enhanced cell functionswere due to the piezoelectric effect rather than the scaffolds composition or thepiezoelectric excitation process. Piezoelectric excitation of the PU/PVDF scaffolds causedthe electrical stimulation, which then enhanced the the cell functions.4. The cells on the implanted scaffolds were mainly fibroblasts. SEM, HE staining andLSCM revealed that the cell activites of the PU/PVDF scaffolds and the PU scaffoldsimplanted in the vertex were almost the same, whereas the cell activites of the PU/PVDFscaffolds were higher than those of the PU scaffolds implanted in the back and abdomen.The enhanced cell activities of the PU/PVDF scaffolds implanted in the back and abdomenwere due to the piezoelectric effect, which was caused by random animal movementsfollowed by mechanical deformation of the scaffolds. Mechanical testing revealed that thefibrosis level of the PU/PVDF scaffolds were higher than that of the PU scaffolds implantedin the back and abdomen. This also indicated that the PU/PVDF scaffolds stimulated cellsand thus increased the fibrosis level.Conclusions1. PU/PVDF scaffolds of different composition ratios were prepared byelectrospinning, among which PU/PVDF (1:1) scaffolds kept a balance between mechanical property and piezoelectric property.2. The micromorphology of the PU/PVDF scaffolds was observed, followed bymeasurement of the fiber diameter and pore size of the scaffolds. Crystalline phasecharacterization confirmed that the PVDF in the scaffolds was mainly in the piezoelectric βphase.3. The fibroblasts cultured on the PU/PVDF scaffolds showed normal morphology andproliferation. The fibroblasts cultured on the piezoelectric-excited scaffolds showedenhanced migration, adhesion and secretion.4. The cell activities of the PU/PVDF scaffolds subcutaneously implanted in SD ratswere enhanced due to the piezoelectrical stimulation, which was caused by random animalmovements followed by mechanical deformation of the scaffolds.5. The scaffolds are potential candidates for wound healing applications.