Fabrication and properties of collagen and polyurethane polymeric nanofibers using electrospinning technique for tissue engineering applications

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 4, Pages: 802-807

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal weblink: http://www.jett.dormaj.com

Fabrication and Properties of Collagen and

Polyurethane Polymeric Nanofibers Using

Electrospinning Technique for Tissue Engineering

Applications

†

† a

Nasrin Beheshtkhoo , Mohammad Amin Jadidi Kouhbanani , Fatemeh sadat Dehghani ,

a,d*

Shahla abdollahii , Mohsen Alishahi , Vahid Razban , Ali Mohammad Amani

Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran

Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shahroud University of Medical Sciences,

Shahroud, Iran

Department of Molecular Medicine, School of Advanced Technologies in MedicineShiraz University of Medical Sciences

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract

The present study introduces a nanofibers skin patch comprised of collagen and polyurethane polymers. Belonging to the family of

biodegradable polymers, they can be mixed with various drugs, degraded at the wounded area, whereby the drug is gradually released

into the wound. The characterization of nanofibers were identified using mechanisms such as SEM, FTIR, and tensile test. The results

of SEM analysis indicated that all the fabricated nanofibers have a proper and uniform morphology. FTIR spectrum for collagen and

TPU revealed various factor groups including N-H group with hydrogen bond, CH2 group, (C=O) carbonyl group, and carboxyl group

(COO) for collagen. Furthermore TPU spectrum demonstrated a broad peak for N-H group, the symmetric and asymmetric stretching

for CH2 group as well as carbonyl group (C=O) peak. In addition, a tensile test examined the mechanical properties of nanofibers with

and without loaded natural honey, indicating that the use of natural honey in the structure of nanofibers decreases the maximum stress

at break point.

Keywords: Nanofiber; Electrospinning; Scaffold; Tissue engineering; Chitosan; Polyurethane



The costs of daily injection may surpass those of topical drug

delivery patch since in the former the drug should be

purchased and injected every day, whereas topical patches

incur less costs due to the continuity and control of drug

delivery process.

In comparison with topical drug delivery, injection drug

delivery is more painful and causes more infectious problems.

In recent years, the use of nanotechnology such as nanofibers

has gained substantial popularity. Nanofibers have enormous

applications in medical sciences including artificial organs,

tissue engineering, medical prostheses, wound dressing, and

drug delivery. Nanofibers are characterized by a unique

ability in loading biological molecules, drugs, and

nanoparticles.

Capable of being fabricated from various substances such

as ceramics and polymers, fibers are thin and long threads

which have high length to diameter ratios. Fibers with

diameters ranging from 1 to 1000 nanometers are called

nanofibers which have different properties including high

surface area to volume ratio, mechanical strength, and

versatility, making them an ideal scaffold for various medical

and engineering applications (1). With regard to medical uses,

nanofibers have special properties such as their similarity to

extracellular matrix (ECM) composed of 10 to over 100

nanometer diameter protein fibers such as collagen. The

Introduction

Skin diseases have been an issue of considerable concern

to the World Health Organization (WHO) such that an

enormous number of studies thus far have been conducted to

find new treatment ways, one of which is to develop an

instrument to topically deliver the drug to the skin. The

advantages of this drug delivery patch over the injection drug

delivery include:

Topical drug delivery to the wound allows the drug to remain

in the injured area in large amounts, inducing less side effects

than injection drug delivery does.

Injection drug delivery has a regular daily basis, that is, the

patient is to receive injection every day, while the topical drug

delivery patch obviates the need for the patient’s presence to

receive injection, making it possible for the patient to replace

the patch after some days.



Corresponding author: Nasrin Beheshtkhoo, Department of

Medical Nanotechnology, School of Advanced Medical

Sciences and Technologies, Shiraz University of Medical

Sciences, Shiraz, Iran.

†

These authors contributed equally to this work.

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 4, Pages: 802-807

relationship between ECM and cells regulates various cell

behaviors including reproduction and gene expression. The

more the resemblance between the fabricated scaffold and

ECM, the better the scaffold can involve in cellular

interactions. Therefore, using nanometer diameter fibers

makes it possible to obtain an appropriate mimic of ECM.

This property turns nanofibers into a unique structure for

medical applications, particularly in tissue engineering (2).

There are various nanofibers fabrication techniques, the

main one being electrospinning method due to its versatility,

adaptability, simplicity and cost-efficiency. Another

advantage of electrospinning technique is its capability in

fabricating fibers from biomaterials such as biodegradable

polymers. When mixed with drugs, polymer solutions turn

into drug-incorporated nanofibers. With degradation of

polymer in the wounded area, the drug is slowly released and

penetrated into the wound (3). Nanofibers can be also utilized

to achieve optimal release rate by controlling various factors

effective on drug release in polymeric matrix structure. It

should be noted that selection of polymer for nanofiber

fabrication is one of the most significant factors (4) as the

polymer should be biocompatible, biodegradable, and have

appropriate physical properties. Depending on the kind of

drug delivery process, releasing drug can be performed in

various durations spanning from several hours to days. Thus,

polymer selection should be in such a way that polymer

degradation rate be proportional to the rate of drug release.

Electrospinning device is made up of four main parts:

collector, but as it approaches the collector, which is

connected to the other end of voltage terminal, the charged jet

whips across space between the needle tip and the collector in

a spiraling way. It is worth noting that the interaction between

various electrospinning parameters should be in a way that the

charged jet maintain a continuous and steady whipping

movement toward the collector.

Fig. 1-1: Taylor cone and tip of needle during electrospinning

Depending on the number of needles, electrospinning is

divided into two uniaxial (single needle) and multiaxial

(multiple needles) processes. In the former, polymeric

solution and the drug are contained in the syringe and are

expelled out toward the collector through a single needle,

whereas in the latter there are two needles as core and shell

and two syringe pumps are used to drive solutions into the

needles (10, 11, 12, 13, 14). Even though electrospinning is a

seemingly simple technique, all the parameters involved such

as the solution, environmental factors, and their interactions

make electrospinning a sensitive process. All in all, they

should interact in such a way that the charged jet steadily

moves toward the collector and that fiber diameters are

consistent and in the expected ranges (15, 16, 17, 18, 19, 20).

A power supply which provides the voltage needed for

electromagnetic force between the needle tip and the

collector.

Syringe pump through which polymeric solution is

pumped

A needle tip connected to one voltage terminal.

A collector connected to other voltage terminal and where

fibers are fabricated

To start electrospinning process, polymeric solution or

melt should be prepared in advance. Then, the polymer

solution is filled in a syringe and placed over the syringe

pump such that a droplet of the polymer solution appears on

the tip of the needle connected to one voltage terminal

Materials and methods

.1 Synthesis

To prepare the solution, various percentages and methods

were used and, in the end, the proper conditions for

fabricating nanofibers without beads were achieved. The

solution was prepared as follows: Thermoplastic polyurethane

(usually negative pole) while the collector is connected to

other terminal (usually positive pole). Given the appropriate

distance between needle tip and the collector screen as well as

the proper applied voltage, the process of nanofiber

fabrication starts (5). After a voltage is applied by the power

supply to the needle tip, this will become highly charged and

the induced surface charges on the polymeric solution will be

evenly distributed over its surface, making the droplet

transform from a rounded (The reason for the rounded droplet

shape is the fact that in the absence of any voltage applied to

the droplet, it tends to form a shape with a less volume to

surface area ratio) to a conical shape (6) also known as

(0.2 gr) was mixed with collagen (0.05 gr) and, then, they

were gradually added to 3.5 ml deionized water and 1.5 ml

hexafluoro-2-propanol (HFIP) while being stirred using a

magnetic mixer at room temperature. After 24 hours, they

were completely dissolved, turning into a colorless, viscous,

uniform and homogeneous solution (21).

.2 Electrospinning setup and specification

The electrospinning setup utilized in the present study was

composed of: a power supply, syringe pump, uniaxial needle

tip, syringe, and a rotating collector To produce the electric

field, a high voltage direct current (DC) power supply was

used. The power supply device (High Voltage 35 OC,

Fanavaran Nano-Meghyas, Tehran, Iran) applied voltages

ranging from 0 to 18 KV. The positive terminal was

connected to the needle tip and the negative terminal to the

rotating collector. Moreover, the syringe pump (model

SP1000HOM, Fanavaran Nano-Meghyas, Tehran, Iran) was

designed to use various kinds of syringes. Given the

dimensions of the syringes, the pump syringe could expel out

a certain amount of solution with the lowest and the highest

“

Taylor cone”, shown in Fig. 1-1. When higher voltages are

applied, the solution is so electrified that it reaches the so

called critical voltage. Consequently, the electric force

overcomes the surface tension of the drop, leading to the

formation of a charged jet which is expelled from the tip of

Taylor cone towards the rotating collector surface where the

fibers are deposited. This cycle continues as the solution in

the syringe pump is charged to expel out more and more

droplets to be changed into fibers (7-8-9).

As the jet stretches out and turns into nanofibers on its

way towards the collector, the solvent evaporates. At the

beginning, the jet moves in a straight line towards the

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 4, Pages: 802-807

speeds of 0.5 µl/h and 1000 ml/h, respectively. The internal

diameter of the syringe was entered into the system using a

keyword, according to which the device computed and

applied the precise amount of solution release. The memory

used in the device was of a permanent type and the last

configurations from syringe size, solution release speed to the

transformation time were recorded in the memory. Also, the

uniaxial needle tip utilized to produce droplets was 0.8192

mm in external diameter (Guage-21 specification). Moreover,

a 5 mm syringe with a 12.5 mm internal diameter was used to

pump the solution into the main syringe. With regard to the

As mentioned before, to perform this tensile test, the fiber

samples were sliced in the form of rectangles with dimensions

of 6×1 cm. They were also placed in a vacuum oven 94 hours

before trial at room temperature and humidity. For this test,

the use was made of Machine Testing Universal Santam

device as well as cell load with the weight of 1.4 kg and

tensile speed of 1 mm/m. The analysis of stress-strain plot

obtained from the device yielded Young’s modulus, tensile

strength and elongation-to-break (24).

2.6 Fourier-Transform Infrared Spectroscopy (FTIR)

In order to identify and analyze the bonds between fibers

and polymers utilized to fabricate nanofibers as well as to

compare their structure, FTIR analysis was employed. FTIR

analysis examines polymers existing in the sample material by

assessing the degree of emission and rotation in bonds of the

material. Given that any material has a certain infrared

spectrum, FTIR is a proper method of confirming the presence

of any bond and of identifying the kind of material being

used. Accordingly, the present study utilized FTIR

spectrometer model RX1 Spectrum manufactured by Elmer

Perkin. To prepare the fibers, they were sliced into small

parts, hence their being formed into tablets and ground along

with potassium bromide (KBr), a neutral salt powder, in front

of infrared radiation (25).

collector, aluminum plates with the dimensions of 25×10 cm

were employed on the roller as the collector and the distance

between the needle tip and the collector was measured

precisely before any trial.(22)

.3 Analysis of Scanning Electron Microscope (SEM)

To examine the morphology and microscopic structure of

nanofibers as well as to determine their diameter, SEM

analysis was conducted on the fibers. To prepare the fibers

for SEM analysis, they were first sliced with dimensions of

×1 cm2 and then mounted on an aluminum foil. In the

present study, we used TESCAN VEGA electron microscope

produced by the Czech Republic. Before imaging, the sliced

sample fibers were covered with gold in 900

angstrom thickness. During SEM analysis, we shine beams of

electrons on the surface of the sample fibers and then the

emissions are scanned while the image of the surface of

samples are shown on a monitor. To better analyze the

morphology of fibers, they were scanned in seven various

zooms

Results and discussion

.1 Nanofiber fabrication results

The present study made an attempt to fabricate

thermoplastic polyurethane (TPU) nanofibers and collagen.

To do so, various electrospinning parameters were initially

evaluated and different concentrations of TPU were prepared

with various injection rates. Afterwards, fiber morphology

and diameter under different electrospinning conditions were

examined via SEM imaging. Such electrospinning parameters

.4 SEM analysis images

In order to determine the diameter of nanofibers, images

from SEM analysis of sample fibers were analyzed using

ImageJ software. To this end, of each sample, a total of 40

fibers were randomly selected and examined. To work by the

software, first certain specified sizes were entered into the

program so as to determine the zoom scale for the software.

Then, the diameter of a fiber was computed by measuring the

distance between the two ends of the fiber. The mean

diameter of measured fibers was reported as the sample fiber

diameter. (23)

should be regulated in

a way that the diameter and

morphology as well as mechanical properties of fibers are

optimized. The parameters for fabricating the optimized

sample fibers were: needle gauge 21, electrospinning solution

with 4% and 3% concentrations of poly-ethylene-oxide (PEO)

and chitosan, respectively. Moreover, the optimal voltage and

the distance between the needle tip and the collector were 10

KV and 11 cm, respectively. The rotating speed of the

collector was also set on 600 rpm along with optimal injection

speed of 0.9 mm/m. Table 3-1- introduces samples fabricated

in various injection rates and chitosan percentages.

.5 Tensile test

In order to assess the mechanical properties of samples,

they were subjected to a tensile test in which sample fibers, in

rectangular form, were stretched out to the point where they

broke in half. In this tensile test, the ultimate plot was a stress-

strain one such that strain was considered sample length per

initial length and stress as force per sample cross section.

Analysis of stress-strain plot gave way to three parameters:

Elastic modulus or Young’s modulus (E), at the beginning of

which there was the slope of stress-strain curve; Tensile

strength also known as the maximum stress in stress-strain

curve; and Elongation-to-break or the degree of strain at the

end of stress-strain curve where the sample broke in halves.

.2 SEM analysis

To examine the morphology, microscopic structure and

diameter of fibers, SEM analysis was performed. To find the

optimal conditions for electrospinning, samples with various

injection rates were analyzed. Fig. 3-1 depicts SEM images

for all the four samples which were analyzed through ImageJ

software so as to compute their mean diameter summarized in

Table 3-2.

Table 3-1: Parameters and amounts used in nanofiber fabrication

Samples

Parameters

TPU (%)

Collagen (%)

Injection Rate (ml/h)

Voltage (KV)

Distance to Collector (cm)

Sample 1

Sample 2

Sample 3

Sample 4

0.5

0.9

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 4, Pages: 802-807

Fig. 3-1: SEM images (from right to left: Sample 1 to Sample 4)

SEM analyses revealed that all the samples had an

appropriate and uniform morphology, according to which

Sample 4, selected as the optimal one, proved to be the most

useful for medical applications (26-27).

elongation-to-break of samples are also depicted by Table 3-3.

Given the results of stress plot on the basis of deformation

percentage as well as the effect of the honey loaded on fibers,

it was concluded that increase in the amounts of honey

decreased maximum stress at break point.

Table 3-2: Mean diameters from SEM images

Samples

Sample 1

Sample 2

Sample 3

Sample 4

Mean Diameter

800

683

917

1003

.3 FTIR results

In this section of the study, various FTIR spectra for

collagen, TPU and synthesized nanofibers were examined.

Regarding collagen FTIR spectrum in peaks 3426, 2940,

660, and 1541, N-H group with hydrogen bond, CH2

3950 3450 2950 2450 1950 1450 950 450

asymmetric stretching, (C=O) carbonyl group stretching,

hydrogen bond coupled with carboxyl (COO), and finally N-

H bending vibration along with C-N stretching vibration were

witnessed. Furthermore, with regard to TPU spectrum, a

broad peak for N-H group was seen in peak 3333, the

symmetric and asymmetric stretching for CH2 group were

respectively witnessed in peaks 2861, and 2934. Also,

carbonyl group (C=O) was witness at peak 1726 with a strong

shoulder near 1700 which was associated with carbonyl

resonance stretching along with urethane-based hydrogen

bonds. Moreover, in areas 1528, and 1100, peaks for N-H

group stretching as well as COC group peak were seen.

According to the comparison of collagen infrared spectra and

TPU, shown by Fig. 3.2 , it was concluded that in the

synthesized nanofiber spectra, all the peaks for collagen and

TPU were witnessed although with a little replacement due to

interactions, suggesting that nanofibers were favorably

synthesized (28, 29, 30, 31) .

Fig. 3-2: FTIR Plot for Synthesized nanofibers

4.5

0.5

10 20 30 40 50 60 70 80 90 100110120130140150

Fig. 3-3: Plot of stress on the basis of nanofiber elongation change

.4 Tensile test

The present study performed a tensile test on sample

fibers so as to examine the effect of loaded drug on the

mechanical properties of fibers. The samples consisted of

optimally electrospun pure fibers composed of natural honey.

The conditions were as follows:

The applied voltage and the distance between needle tip and

the collector were 10 KV and 11 cm, respectively. The stress

plot of the fibers on the basis of samples deformation

percentage is shown by Figs. 3-3 and 3-4. Also, the stress and

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 4, Pages: 802-807

Conclusion

Sseveral studies have been carried out on syntheses of

nanoparticles (41–50). The present study was carried out with

the aim of examining the fabrication and properties of

collagen and polyurethane polymeric nanofibers using

electrospinning technique. SEM technique was employed to

investigate the morphology, microscopic structure as well as

diameter of nanofibers, as a result of which the best nanofiber

for medical applications was selected. Afterwards, the optimal

nanofiber was chosen using FTIR analysis. To do so, a

comparison of IR spectrum of raw material and the product

helped us come to the conclusion that the nanofiber was

favorably synthesized. In the end, we used tensile test to

examine mechanical properties of the selected nanofiber

loaded with natural honey as the delivered drug, suggesting

that the nanofiber had optimal tensile properties. Also, it was

found that using natural honey in the structure of nanofiber

decreased the maximum stress at break point.

10 20 30 40 50 60 70 80 9010 01 1 01 2 01 3 01 4 01 5 01 60

Fig. 3-4: Plot of stress on the basis of honey-loaded nanofiber

elongation change

Table 3-3: Mechanical properties of nanofibers with various concentrations of loaded drug

Sample

Stress at break point

Elongation-to-break

142.04

Nanofibers without natural honey

Nanofibers with natural honey

4.70

3.01

153.07

Nanotechnology 17.14 (2006): R89.

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