Electrospun Core-Sheath Bicomponent Nanostructures for Soft Tissue Engineering

NTC Project: F05-NS04

National Textile Center Annual Report: November 2008

1

Electrospun Core-Sheath Fibers for Soft Tissue Engineering

Project No.: F05-NS04

Project Team:

Leader: Bhupender S. Gupta/ NC State University

[email protected]

Members: Martin W. King/ NC State University

Samuel Hudson/ NC State University

Elizabeth G. Loboa/ NC State University

Rudolf Hufenus/ Post-doctoral Fellow

Jessica Gluck/ Graduate Student

Carla Haslauer/ Graduate Student

Venugopal Boppa/ Graduate Student

Ajit Moghe/ Graduate Student

GOAL STATEMENT

The goal of this research is to develop novel bi-component nanofiber structures, using natural

and synthetic biodegradable polymers that could be used as efficient scaffolds for engineering

soft tissues.

ABSTRACT

The current project involves co-axial electrospinning of two different polymers, one natural and

the other synthetic, both biodegradable, to produce ‘sheath-core’ structures. The project, in the

first phase, focused on the development of the technology required for the production of uniform

core-sheath fibers and on the determination of the factors that affected the morphology of the

fibers produced. The required coaxial spinning system set-up involved two syringes with needles

and two pumps to deliver the solutions in predetermined rates. For this phase, the polymers used

were polyvinyl alcohol (PVA) and polyethylene oxide (PEO). The TEM studies of the cross-

sections of the fibers clearly revealed the core-sheath structure. In the second phase of this

project after achieving the success in the approach used, the desired combinations of the natural

and synthetic biodegradable polymers were selected and the effects of sheath and core polymer

concentrations and feeding rates were determined. The first set consisted of gelatin as the sheath

and polycaprolactone (PCL) as the core. TEM studies, as well as SEM examinations after freeze-

fracturing, demonstrated the presence of the core-sheath bicomponent morphology in the fibers.

The second set of polymers, suitable for actual tissue engineering applications, consisted of

collagen as the sheath and polycaprolactone (PCL) as the core. This set was selected with the

hypothesis that the collagen sheath will aid in cell adhesion and proliferation whereas PCL core

will impart strength and elasticity over an extended period. After initiating cell growth, collagen

will biodegrade exposing the PCL core, which will provide mechanical and dimensional stability

to the developing tissue as it continues to mature to its final stage. By this time, PCL will have

degraded, leaving the engineered tissue ready for implantation.

mailto:[email protected]



2

The effects of solution concentration, flow rate, and applied voltage on the dimensions and the

morphology of the bicomponent fibers were investigated. Following this investigation, the

degradation of the collagen-PCL fibers was studied. This behavior was modeled to predict the

degradation rate of collagen in the sheath. The cell culture studies were performed to determine

the efficacy of the structure to support the cell growth. The results demonstrated that the use of

collagen in the sheath enhanced the cell spreading and differentiation behaviors. Immediate

future plan includes the assessment and modeling of the mechanical properties of the structures.

BACKGROUND AND INTRODUCTION

The demand for novel nanostructures in areas such as biotechnology, telecommunications and

computing has increased enormously in recent years1,2

. In particular, electrospun nanofiber

structures, using natural or synthetic biodegradable polymers, have drawn increased interest for

use as scaffolds for tissue engineering. Electrospun nonwoven webs containing sub-micron

diameter fibers serve as near ideal substrates to grow soft tissues in that they provide a unique

structure characterized by a high surface area to volume ratio and three-dimensional

interconnected pore network, both of which enhance cell attachment and proliferation3.

Additionally, it is widely accepted that the electrospinning technique can lead to structures that

resemble native extra-cellular matrix (ECM) elements4. Cell -culture studies on such materials

have demonstrated that the scaffolds become densely populated with cells in a short time and

they promote cellular infiltration into the fibrillar network5. It has also been shown that the cell

adhesion and proliferation rate significantly improves on nanofibrous scaffolds as compared to

plane porous polymer films6.

To optimize tissue engineering potential of the scaffold, it is necessary that the latter (1) has cell-

binding capabilities to allow cells to adhere and proliferate, (2) has adequate strength to support

the developing tissue, and (3) biodegrades with time to allow more cells to fill the volume and

the engineered tissue, without the scaffold, to become the implant. An optimum structure thought

of is a biodegradable bicomponent fiber with the sheath of natural and the core of synthetic

polymers.

The primary reason for using natural polymers is that they are inherently capable of binding cells

since they carry specific protein sequences, such as RGD (arginine/ glycine/ aspartic acid)7.

Synthetic biodegradable polymers provide the necessary mechanical properties, such as

viscoelasticity and strength, and their degradation rate can be controlled as needed8.

Accordingly, there is merit in combining natural and synthetic polymers to produce materials

that have novel hybrid properties at the sub-micron level. Such composite structures using

different combinations of polymers may prove to be valuable for a variety of other applications

as well, such as for drug delivery, optical fiber, and circuit board nanocables systems.

A novel idea in developing scaffolds is to use a core-sheath structure with two different polymers

that degrade at different rates. Such spinning was first demonstrated by King et al. using

bicomponent carpet fiber melt-spinning technology to spin resorbable materials9. The idea of co-

axial electrospraying for encapsulation of liquid droplets was introduced by Loscertales et al10

and has been successfully applied to electrospinning of composite and hollow fibers by several

groups11-15

. For the current work, a similar system was set up for electrospinning of co-axial

bicomponent nanofibers using biodegradable polymers.



3

PROGRESS TO DATE

Co-axial needle set-up and co-electrospinning process:

The system consists of two syringes (Figure 1). A long needle connected to the back syringe and

a short needle connected to the front syringe make the ‘co-axial’ configuration at the tip. The

front syringe contains the sheath polymer solution while the back holds the core polymer

solution. The syringes are independently driven to control the flow rates of the two polymer

solutions.

Figure 1: Co-axial electrospinning system and the process

The remainder of the system includes the components commonly used for electrospinning

processes: these are the grounded collector plate and the high voltage power supply connected

between the needle and the collector.

When the voltage is gradually increased, the composite droplet formed at the tip of the needle

undergoes transformation in shape to form the ‘Taylor cone’16

. Once the power reaches a certain

threshold value, a jet containing the two polymers emerges from the vertex of the cone and

undergoes whipping instability to form nanometer sized fibers11,17

. Solvent evaporates in the

process and dry fibers are collected on the collector plate in the form of a non-woven mat.

Initial studies:

Preliminary studies to determine the feasibility of the system to produce bicomponent nanofibers

were performed using polyvinyl alcohol (PVA) and polyethylene Oxide (PEO). Solutions with

different concentrations of PVA were prepared using deionized water as a solvent. PEO, on the

other hand, was dissolved in deionized water as well as in chloroform to form solutions of

different concentrations. A small amount of dye was added to the solution made with chloroform

to observe the relative position of the components. Different solvents were used to observe the

effect of the combination on the core-sheath distribution of the polymers. Prior to conducting the

conjugate spinning, the individual polymers were electrospun to determine the optimum

conditions (polymer concentration, flow rate, and voltage) needed to make uniform fibers. These

conditions were then applied to prepare the bicomponent fibers.

Co-axial Jet

Whipping Jet

Collector

High Voltage Supply

Sheath Solution

Core Solution



4

The characterization of the electrospun mats was performed using electron microscopy

techniques. The morphology was examined under SEM and the cross-sections of the fibers were

observed under TEM. Ultramicrotomy was performed to obtain sections approximately 100 nm

in thickness. The preliminary results, shown in Figure 2, clearly reveal the core-sheath

morphology in the electrospun nanofibers.

Figure 2: TEM cross-sections of PVA-PEO (Sheath-Core) Bicomponent Fibers

Electrospinning of target polymers:

After evaluating the feasibility of the approach, the desired combinations of natural and synthetic

biodegradable polymers were selected. The first set consisted of gelatin as the sheath and

polycaprolactone (PCL) as the core. Both polymers were dissolved in glacial acetic acid as the

solvent. The structure of the electrospun fibers produced was characterized using electron

microscopy techniques. The cross-sections examined under TEM clearly revealed the core-

sheath morphology in the fibers (Figure 3).

Figure 3: Evidence of core-sheath structure (TEM, nanofibers: Sheath, Gelatin; Core, PCL)

Another technique, i.e. freeze fracturing, was employed to induce differential fibrillation in the

sheath and core. The fiber subjected to this treatment also gave the evidence of the presence of

core-sheath arrangement of the two components (Figure 4).

Figure 4: FE-SEM of freeze-fractured nanofibers: Sheath, Gelatin; Core, PCL)



5

The second and final set of polymers, more suited for actual tissue engineering application,

consisted of collagen as the sheath and polycaprolactone (PCL) as the core. For collagen/ PCL

combination, both polymers were dissolved in 1,1,1,3,3,3- Hexafluoroisopropanol (HFIP).

Freeze-fracturing and subsequent viewing under SEM yielded images that proved the core-

sheath morphology of fibers generated from this set of polymers (Figure 5).

Figure 5: Evidence of core-sheath structure (FE-SEM views of the freeze-fractured nanofibers-

Sheath: Collagen, Core: PCL)

After the successful electrospinning of the target polymers, the effect of processing parameters

(applied voltage, flow rates, and polymer concentrations) on fiber size and morphology were

examined. The objectives were to identify conditions that led to uniform structures and the

criteria one could use to develop structures with specific morphologies (core, sheath, and overall

fiber thicknesses) for each different tissue engineering application.

Effect of applied voltage

For a given pair of polymer systems and flow rates, it was found that there existed a narrow

range of applied voltage in which a stable compound Taylor cone formed (Figure 6B). Voltage

below the critical range caused dripping of the two solutions followed by an intermittent jet from

the sheath (Figure 6A) with an occasional incorporation of the core. Also, due to increased size

of the cone, mixing of the two solutions tended to occur. Voltage above the critical range caused

the strength of the electric field to exceed that required for the material and the processing

conditions used. This led the Taylor cones to recede into and the jets to emerge from inside the

capillaries. Instead of the co-axial jet, separate jets formed from the sheath and core solutions

(Figure 6C). As expected, the structure obtained showed high variability in terms of fiber

diameters (Figure 7), which suggested that different fibers formed from the sheath and the core

solutions.

Figure 6: Schematic of the voltage dependence of the core-sheath fiber formation in co-axial

electrospinning (A: Subcritical voltage; B: Critical voltage; C: Supercritical voltage

B C A

Increasing voltage



6

Figure 7: Effect of applied voltage on fiber morphology, A: Critical voltage, B: Higher voltage

The critical voltage in which optimum spinning occurred was found to lie in a narrow range (as

small as 1 kV for the gap of 15 cm between the capillary and the collector). The optimum value

and the range in which it lay were needed to be determined experimentally for each different pair

of solutions (Table 1).

Table 1: Critical voltage range observed for the two polymer combinations

Polymers Critical voltage range

Collagen/PCL 9 – 10 kV

Gelatin/ PCL 10.5 – 11.5 kV

Effect of solution concentrations and flow rates

Flow rates used for the polymers showed a positive effect on the fiber diameter. The average

fiber diameter increased from 325 60 nm to 492 115 nm on increasing the flow rate from 150

l/ hr to 300 l/ hr, respectively (Figure 8). Since the rates for the sheath and the core solutions

were the same, this increase in diameter implied that both the sheath thickness and the core

diameter increased due to more materials delivered at a given time.

The concentration of polymer also affected the fiber size. Overall, the fiber diameter increased

from 232 40 nm to 492 115 nm on increasing gelatin concentration in the sheath from 9 to 12

%, respectively (Figure 9A). This suggested that the sheath thickness increased due to the

presence of more material in the solution. Similarly, an increase in the PCL concentration in the

core from 10 to 12 %, the fiber diameter increased from 254 44 nm to 310 49 nm,

respectively. The increase in diameter, in this case, indicated that the core size increased (Figure

9B).

[A]

[B]



7

Figure 8: Effect of solution flow rate on the nanofiber diameter (Sheath: Gelatin; Core: PCL)

Figure 9: Effect of solution concentration on the nanofiber diameter. A: effect of sheath polymer

concentration (Sheath: Gelatin; Core: PCL), B: effect of core polymer concentration (Sheath:

Collagen; Core: PCL)

In-vitro hydrolytic degradation studies:

Prepared collagen-PCL structures were subsequently examined for their in vitro hydrolytic

degradation behavior in PBS solution. The degradation behavior was characterized using

morphological, weight loss, spectrographical, and elemental analyses.

Figure 10: Weight-loss behavior of the collagen-PCL sample with respect to time

150 l/ hr 300 l/ hr

10 % 12 %

B

9 %

12 %

A



8

Owing to degradation, the fiber diameters for the sample decreased significantly (from 418 nm to

360 nm) within the first hour. As expected, the weight loss of the fibers was time dependent

(Figure 10). It was highest within the first hour, after which the degradation was slow and

gradual. It was established from the elemental and spectrographical analyses that the

degradation only appeared to take place in the collagen sheath and not in the PCL core. This

behavior was modeled using a mathematical approach. The model suggested that the mass loss

of collagen was proportional to the natural logarithm of degradation time. At the end of the thirty

day period of observation, although collagen had lost most of its mass (about 85%), the sample

retained its structural integrity due to the presence of PCL in the core.

Cell-culture Studies:

Collagen-PCL bicomponent fibers were characterized for their ability to support cell growth for

use in tissue engineering applications. Human adipose derived adult stem cells (hADASCs) were

used for this purpose. The cell growth behavior on the scaffolds was characterized at different

time intervals (24, 48, 72 hrs, 1week, and 2 weeks) using cell viability and cell differentiation

assays.

Cell viability assessed the effectiveness of the collagen-PCL and PCL (control) scaffolds to

support the cell survival. In this assay, it was found that the majority of the cells were alive on

both scaffolds, but during the first week of culture the cells spread out differently on the two

surfaces. On collagen-PCL scaffolds, cells occupied the entire surface uniformly; on PCL

control, in contrast, the cells were concentrated in segregated areas. This behavior was attributed

to the hydrophobicity of PCL which prevented the surface from wetting by the culture media

and, therefore, led to cells being present nonuniformly over the area. After 1 week, however, the

cells spread out evenly on the PCL scaffold surfaces as a result of increased hydrophilicity. This

presumably resulted from the adsorption of the proteins from the culture media during the first

week of culture.

The potential of the scaffolds to support cell differentiation in osteogenic pathway was

characterized using the cell differentiation assay. It was found that the differentiation was about

five times more on the collagen-PCL bicomponent scaffolds than that on the PCL mono-

component structures (Figure 11).

Figure 11: Cell differentiation of the collagen-PCL and the PCL control scaffolds.



9

Cells produce calcium as they differentiate from the stem cells into the bone cells. Therefore, this

change was characterized by measuring the calcium content in the scaffolds. The results clearly

indicated that the presence of collagen in the sheath improved the biocompatibility of the

scaffold structure and hence, enhanced the ability of the stem cells to differentiate into the bone

cells.

SUMMARY AND CURRENT STATUS

Using electrospinning, core-sheath nanofiber structures containing two different combinations of

natural and synthetic biodegradable polymers were successfully prepared. It was observed that

the applied voltage used played an important role in achieving the core-sheath bicomponent

morphology during the process. For developing uniform core-sheath structures, the applied

voltage required generally lied in a narrow range, which was needed to be identified

experimentally for each pair. Polymer solution flow rates and concentrations were found to

directly control the core-sheath fractions and, consequently, the overall nanofiber diameter. In-

vitro degradation studies were performed on Collagen-PCL bicomponent nanofibers. The results

showed that only collagen from the sheath degraded within the observed period and, as

anticipated, the use of synthetic polymer (PCL) into the core improved the mechanical stability

of the biodegradable structure. Cell culture studies on collagen-PCL structures clearly illustrated,

as hypothesized, the superior performance of bicomponent scaffolds over that of

monocomponent PCL controls.

After determining the efficacy of the bicomponent fibers for tissue engineering, the focus of the

current work has been to study and analyze the mechanical properties of the electrospun webs as

these properties are critical for supporting the long term cell growth activity. In the first part of

this phase, our focus has been on determining the effects of fiber diameter and fiber degradation

on the mechanical properties. In the second and final part, our aim will be to model these

behaviors in order to predict the mechanical properties of these structures. The experimental and

the theoretical work related to mechanical properties is currently in progress18-21

.

ACKNOWLEDGEMENTS

The principle investigator and the members of the committee wish to thank the National Textile

Center for providing funding and the opportunity to work on this project.

PROJECT WEBSITE ADDRESS

http://www.ntcresearch.org/projectapp/?project=F05-NS04

REFERENCES:

1. Bashir R. DNA-mediated artificial nanobiostructures: state of the art and future

directions. Superlattices and Microstructures 2001;29(1):1-16.

2. Tegart G. Nanotechnology: The Technology for the 21st Century. The 2nd International

Conference on Technology Foresight - Tokyo, 27-28 Feb. 2003.

http://www.ntcresearch.org/projectapp/?project=F05-NS04



10

3. Yashimoto H, Shin YM, H.Terai, Vacanti JP. A biodegradable nanofiber scaffold by

electrospinning and its potential for bone tissue engineering. Biomaterials

2003;24(12):2077-2082.

4. Wnek GE, Carr ME, Simpson DG, Bowlin GL. Electrospinning of nanofiber fibrinogen

structures. Nano Letters 2003;3(2):213-216.

5. Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of Collagen

Nanofibers. Biomacromolecules 2002;3:232-238.

6. Xu CY, Inai R, Kotaki M, Ramakrishna S. Aligned biodegradable nanofibrous structure:

a potential scaffold for blood vessel engineering. Biomaterials 2004;25:877-886.

7. Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated

by small synthetic fragments of he molecule. Nature 1984;309:30-33.

8. Hakkarainen M. Aliphatic Polyesters: Abiotic and Biotic Degradation and Degradation

Products. Adv. Polym. Sci. 2002;157:113-138.

9. King MW, Ornberg RL, Marois Y, Marinov GR, Cadi R, Southern JH, S.J. J, Weinberg

SL, Shalaby SW, Guidoin R. Partially Bioresorbable Bicomponent Fibers for Tissue

Engineering: Mechanical Stability of Core Polymers. Trans. 6th World Biomaterials

Congress 2000:533.

10. Loscertales IG, Barrero A, Guerrero I, Cortijo R, Marquez M, Ganan-Calvo AM.

Micro/Nano Encapsulation via Electrified Coaxial Liquid Jets. Science 2002;295:1695-

1698.

11. Sun Z, Zussman E, Yarin AL, Wendorff JH, Greiner A. Compound Core-Shell Polymer

Nanofibers by Co-Electrospinning. Advanced Materials 2003;15(22):1929-1932.

12. Loscertales IG, Barrero A, Márquez M, Spretz R, Velarde-Ortiz R, Larsen G. Electrically

Forced Coaxial Nanojets for One-Step Hollow Nanofiber Design. J. Am. Chem. Soc.

2004;126 (17):5376- 5377.

13. Li D, Babel A, Jenekhe SA, Xia Y. Nanofibers of Conjugated Polymers Prepared by

Electrospinning with a Two-Capillary Spinneret. Advanced Materials 2004;16(22):2062-

2066.

14. Yu JH, Fridrikh SV, Rutledge GC. Production of Submicrometer Diameter Fibers by

Two-Fluid Electrospinning. Advanced Materials 2004;16(17):1562-1566.

15. Díaz JE, Loscertales IG, Lallave M, Galán D, Márquez M, Barrero A. Core-shell

Nanofibers in One Step from Electrified Jets. The Fiber Society Book of Abstracts, 2005

Spring Conference, St. Gallen, Switzerland 2005:46.

16. Reneker DH, Chun I. Nanometer diameter fibers of polymer, produced by

electrospinning. Nanotechnology 1996;7:216-223.

17. Shin YM, Hohman MM, Brenner MP, Rutledge GC. Electrospinning: A whipping fluid

jet generates submicron polymer fibers. Applied Physics Letters 2001;78(8):1149-1151.

18. J W S Hearle and P J Stevenson, ‘Studies in nonwoven fabrics. Part IV: Prediction of

tensile properties,’ Textile Res. J., 34(3), 1964, 181-191

19. J W S Hearle and P J Stevenson, ‘Nonwoven fabric studies. Part XIV: Derivation of

generalized mechanics by the Energy method,’ Textile Res. J., 37(9), 1967, 778-797

20. J W S Hearle and P J Stevenson, ‘Nonwoven fabric studies. Part XV: The application of

the fiber network theory,’ Textile Res. J., 38(4), 1968, 343-351

21. A K Paradkar, ‘Structural mechanics of nonwoven fiber web,’ M.S Thesis, North

Carolina State University, 1987

Electrospun Core-Sheath Bicomponent Nanostructures for Soft Tissue Engineering

Documents

Transcript of Electrospun Core-Sheath Bicomponent Nanostructures for Soft Tissue Engineering