Effects of Processing Parameters on the Morphology and Size of

Electrospun PHBV Micro- and Nano-fibers

Ho-Wang Tong1, Min Wang2

Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong

1boris-tong@hkusua.hku.hk,

2memwang@hku.hk

Keywords: Electrospinning, PHBV, Nanofiber, Microfiber, Tissue Engineering, Scaffold

Abstract. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was used to fabricate micro- and

nano-fibrous, non-woven mats by electrospinning for potential tissue engineering applications. The

morphology and size of electrospun fibers were assessed systematically by varying the processing

parameters. It was found that the diameter of the fibers produced generally increased with

electrospinning voltage, needle diameter for the polymer jet and polymer solution concentration.

Beaded fibers were readily produced at low PHBV concentrations, whereas the needle was blocked

within a very short time during electrospinning when the PHBV concentration was too high. At the

polymer concentration of 7.5 % w/v, it was shown that beadless PHBV fibers could be generated

continuously by adjusting the electrospinning parameters to appropriate values. This study has clearly

demonstrated that electrospinning can be an effective technique to produce PHBV micro- and

nano-fibers. It has also been shown that composite fibers containing hydroxyapatite (HA) can be

produced using the electrospinning technique.

Introduction

Tissue engineering offers a promising new means for repairing human body tissues that cannot be

regenerated on their own after trauma or disease. Restoration of tissue functions by utilizing tissue

engineering techniques usually requires the use of a scaffold.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), in addition to being a natural polymer which

is biocompatible and inexpensive, exhibits complete biodegradability without any toxic byproducts.

Such a polymer has the potential of being a good material for constructing tissue engineering

scaffolds [1, 2]. On the other hand, researchers have found that cells attach to and organize well

around fibers with diameters smaller than the diameter of the cells [3, 4]. Therefore, it is advantageous

to construct micro- and nano-fibrous scaffolds for tissue engineering. Electrospinning, a technology

capable of producing fibers with diameters ranging from nanometers to micrometers, is an attractive

scaffold manufacturing method. The morphology and size control of electrospun fibers for tissue

engineering is important because fiber diameter affects cell behaviour. Furthermore, formation of

beads on electrospun fibers decreases the surface area-to-volume ratio significantly, which in turn

decreases the degradation rate of fibers. Hence it is essential to generate bead-free fibers of

appropriate diameters. Therefore, a systematic study was performed on the electrospinning of PHBV

fibers in order to investigate the effects of electrospinning parameters. In addition, the feasibility of

producing composite fibers containing PHBV and hydroxyapatite (HA) was assessed.

Materials and Methods

PHBV containing 2.9 mol% of 3-hydroxyvalerate and having a molecular weight of 310,000 was

commercially available (Tianan Biologic Material Ltd., China). The solvent chloroform was

analytical grade. The polymer and solvent were used in the as-received state without further

purification. To prepare polymer solutions for electrospinning, PHBV was dissolved in chloroform

using a hotplate magnetic stirrer. Different PHBV concentrations from 4.5 to 25 % w/v were

prepared. The experimental setup for electrospinning in this study is shown schematically in Fig. 1.

Key Engineering Materials Vols. 334-335 (2007) pp 1233-1236Online available since 2007/Mar/15 at www.scientific.net© (2007) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/KEM.334-335.1233

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The polymer solution was pushed from a syringe to a needle via a silicone rubber tube. The solution

feeding rate, which was varied from 1 to 9 ml/h in this study, was controlled by a syringe pump.

Needles with inner diameters varying from 0.4 to 1.2 mm were used. High voltage, varying from 5 kV

to 35 kV, was applied to the needle by a high voltage power supply (Gamma High Voltage Research,

USA). A grounded collector made of an aluminium foil was placed below the needle. The distance

between the needle and the collector (i.e., the working distance) was varied from 12.5 to 30 cm. Due

to the high electrical potential difference between the needle and the collector, the polymer solution

was ejected from the needle towards the collector as shown in Fig. 2. As the polymer jet traveled in

air, the solvent evaporated and the polymer solidified into ultra fine fibers which were collected as a

non-woven fiber mat on the aluminium foil. The electrospun PHBV fibrous mats were then kept

under vacuum for 2 days at ambient temperature in order to remove the solvent completely. The

morphology of the electrospun fibers was observed using scanning electron microscopy (SEM,

Stereoscan 440, UK). The fiber diameter was measured by analyzing SEM images of fibers through

an image analysis program. The diameters of fifty individual fibers were measured and the fiber

diameters were then averaged. To produce bioactive scaffolds for bone tissue engineering, nano-sized

HA particles produced in-house [5] were used.

Fig. 1: The setup for electrospinning of fibers Fig. 2: The start of electrospinning

Results and Discussion

To study the effects of polymer solution feeding rate, all parameters except the feeding rate were

fixed. It was found that the average diameter of the PHBV fibers was about 1.4 µm when the solution

feeding rate was varied between 1 ml/h and 7 ml/h. Beaded fibers were generated when the solution

feeding rate became 9 ml/h. The effects of needle diameter were investigated by using needles of

different sizes while other parameters were unchanged. The average diameter of electrospun fibers

increased from 2.38 µm to 5.56 µm when the needle diameter was increased from 0.4 mm to 0.7 mm

(Fig. 3a). Further increase in needle diameter resulted in beaded fibers. The effects of electrospinning

voltage, polymer solution concentration and working distance were assessed in a similar way. The

average fiber diameter increased from 3.53 µm at 20 kV to 5.41 µm at 35 kV electrospinning voltage.

Beaded fibers were formed when the voltage was lower than 20 kV. The average fiber diameter

increased from 1.35 µm when the polymer solution concentration was 7.5 % w/v to 3.3 µm when the

solution concentration was 25 % w/v. Only beaded fibers were formed at the solution concentration of

4.5 % w/v while clogging easily occurred at the tip of the injection needle when the polymer

concentration was above 25 % w/v. In contrast, the average fiber diameter decreased from 4.51 µm to

2.02 µm as the working distance increased from 12.5 cm to 22.5 cm (Fig. 3b). Further increase in

Rubber tube Syringe

Syringe pump

High voltage

power supply Aluminium foil

Needle tip

without

voltage

Pendent droplet

Initial jet

Taylor cone

Needle tip

with high

voltage Needle

1234 Advances in Composite Materials and Structures

working distance above 22.5 cm generated beaded fibers. Beadless and beaded fibers are shown in

Fig. 4 and Fig. 5, respectively.

(a) effect of electrospinning needle diameter (b) effect of working distance

Fig. 3: Effects of processing parameters on the average diameter of PHBV fibers

Fig. 4: SEM micrograph of beadless fibers Fig. 5: SEM micrograph of beaded fibers

(a) (b)

Fig. 6: HA/PHBV composite fibers: (a) SEM micrograph, and (b) EDX spectrum of the area marked

in (a) indicating the presence of HA particles

Generally, the proper in vivo phenotype cannot be achieved consistently if cells are presented with

fibers having diameters equal to or an order of magnitude greater than the cell size [6]. Unfortunately,

typical scaffold strut diameters approach 10 µm, which is comparable to the diameter of a cell.

Constituents of the natural extracellular matrix (ECM) exhibit fiber diameters from 50 nm to 150 nm,

which are far smaller than the typical scaffold strut diameters [7]. It was shown in the current

Key Engineering Materials Vols. 334-335 1235

investigation that electrospinning could successfully produce PHBV fibers having an average

diameter down to 1.26 µm, which approaches the natural template. For tissue engineering scaffold

applications, different fiber diameters are required depending on the tissue [8]. Therefore, the current

investigation also focused on how the size of electrospun fibers could be controlled by processing

parameters. The findings obtained in this study are useful for electrospinning PHBV fibrous scaffolds

targeted for different tissues. The mechanical strength is another important parameter in scaffold

design, especially for bone tissue engineering. The compressive strength of a tissue engineering

scaffold can be increased by incorporating bioceramic particles in the polymer scaffold. Furthermore,

the incorporation of bioactive bioceramic such as HA into polymer scaffolds should enhance

osteoconductivity of the scaffolds. In the current investigation, by dispersing HA particles in the

PHBV solution, HA particles could be encapsulated in the electrospun PHBV fibers. The feasibility

of HA incorporation was supported by SEM and energy-dispersive X-ray spectroscopy (EDX) results

shown in Fig. 6. It was found that EDX spectra of composite fibers exhibited Ca and P peaks,

confirming the presence of HA particles inside the fiber. Further investigations are needed to optimize

the electrospinning parameters for composite fibers.

Conclusions

The electrospinnning technique has been successfully employed to fabricate PHBV micro- and

nano-fibers. The fiber diameter generally increased with polymer solution concentration,

electrospinning voltage and injection needle diameter and decreased with working distance.

Appropriate adjustment of the processing parameters could generate ultra-fine fibers without beads. It

is also feasible to electrospin composite fibers containing nano-sized bioceramics such as HA. The

composite fibers should be useful for bone tissue engineering.

Acknowledgements

This work was supported by the Nano-biotechnology Strategic Research Theme of the University of

Hong Kong and a CERG grant (HKU 7182/05E) from the Research Grants Council of Hong Kong.

H.W.Tong thanks the University of Hong Kong for providing a research studentship.

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