graphene oxide-decorated plga/collagen hybrid fiber sheets for application to tissue ...1)...

Biomaterials

Research

C The Korean Society for Biomaterials

Biomater. Res. (2014) 18(1) : 18-24

18

Graphene Oxide-decorated PLGA/Collagen Hybrid Fiber Sheets forApplication to Tissue Engineering Scaffolds

Eun Ji Lee1†, Jong Ho Lee1†, Yong Cheol Shin1, Dong-Gook Hwang2, Jin Soo Kim2, Oh Seong Jin1,Linhua Jin1, Suck Won Hong1, and Dong-Wook Han1,2*

1Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735, Korea2Department of Applied Nanoscience, College of Nanoscience & Nanotechnology, Pusan National University, Busan 609-735, Korea

(received August 20, 2013 / revised October 11, 2013 / accepted October 15, 2013)

In this study, novel graphene oxide (GO)-decorated hybrid fiber sheets composed of poly(lactic-co-glycolic acid,PLGA) and collagen (Col) (GO-PLGA/Col) for application to tissue engineering scaffolds were prepared via dual elec-trospinning. Physicochemical properties of GO-PLGA/Col fiber sheets were characterized by field emission scanningelectron microscopy (FESEM), atomic force microscopy (AFM), Fourier transform infrared (FTIR) and Raman spec-troscopy, thermogravimetric analysis (TGA) and contact angle measurement. FESEM and AFM images showed thatGO-PLGA/Col fiber sheets had a three-dimensional interconnected pore structure with an average fiber diameter ofabout 480 nm. FTIR and Raman spectra revealed that GO was uniformly distributed in the fiber structure of PLGAor PLGA/Col sheets. TGA profiles demonstrated that GO-PLGA/Col hybrid fiber sheets were thermally stable in spiteof adding GO. GO slightly affected the contact angle of PLGA sheets, while Col significantly increased their hydro-philicity. Initial attachment of human dermal fibroblasts (HDFs) on GO-PLGA and GO-PLGA/Col fiber sheets wassignificantly superior to that on PLGA sheets, and their proliferation was gradually increased during the cultureperiod. These results suggest that GO-PLGA/Col hybrid fiber sheets can be effectively used as scaffolds supportingtissue regeneration.

Key words: graphene oxide, PLGA, collagen, fiber sheets, tissue engineering

Introduction

ffective wound healing involves a complicated interaction

between epidermal and dermal cells, the extracellular

matrix (ECM), and angiogenesis; all regulated by an array of

cytokines and growth factors.1) Tissue engineering has emerged

as a promising approach to treat the loss or defective wound

area, thereby improving wound healing process. Such an

approach includes scaffolds, cells and biological factors alone

or in combination.2) There are many researches towards

employing electrospinning for scaffold fabrication because the

mechanical, biological and physicochemical properties of the

scaffold are easily adjusted by alteirng the polymer solution

composition and processing parameters. Also, electrospinnig

method have the ability to produce a non-woven nano-/

micro-fibrous structure, which has a morphological and archi-

tectural similarity to the ECM of natural tissue.3) The electro-

spun fibers have some features including high surface-to-

volume ratio, microporous structure, afford quickly start signal-

ing pathway and spatial interconnectivity, which are well-suited

for nutrient and waste transport and cell communication.4,5)

The hybrid fiber sheets we present in this paper are dual-

electrospun two population of fibers, collagen (Col) and poly

(lactic-co-glycolic acid) (PLGA), where the PLGA fibers were

decorated directly with the graphene oxide (GO). Col is the

most abundant protein in the human body, where it conducts

as an essential structural and mechanical building block of the

extracellular matrix in all the tissue.6) Col has many great

properties to be utilized as tissue engineering scaffolds such as

biocompatibility, non-immunogenicity and bioresorbility. Tai-

lored ultra thin collageneous sheets have been electrospun to

prepare synthetic ECM for tissue-engineering scaffolding.7,8)

However, the use of Col alone is limited due to its poor

mechanical properties and rapid degradation behaviors.9)

PLGA is a family of FDA-approved biodegradable synthetic

polymers for manufacturing electrospun fiber. As compolymers

of PLA and PGA, PLGA is physically strong and highly bio-

compatible and has an amenability to modification.10,11) PLGA

is most popular among the various available biodegradable

polymers because of its long clinical experience, favorable

degradation characteristics which can be easily tailored by

E

†These authors contributed equally to this work.*Corresponding author: [email protected]

Graphene Oxide-decorated PLGA/Collagen Hybrid Fiber Sheets for Application to Tissue Engineering Scaffolds 19

Vol. 18, No. 1

altering the lactide/glycolide ratio and by their molecular

weight.12,13) Graphene is a carbon crystal with a two-dimen-

sional, honeycomb-lattice structure. It has extraordinary physi-

cal and chemical properties including high electrical and

thermal conductivities, exceptional mechanical strength and

stiffness, distinctive optical properties, and extreme chemical

stability.14,15) GO ,which is the most popular chemically modi-

fied graphene, is synthesized by a variety of strong chemical

oxidants. GO still retains a layered structure, but it is much

lighter in color than graphene due to the loss of electronic

conjugation brought about by the oxidation.16,17) And recently,

Graphene and GO have attracted attention in biological and

biotechnical application such as scaffold substrates for tissue

regeneration, carriers for drug or gene delivery, detection

materials for bioimaging and bio-sensors, and neural inter-

faces.18-20) Above all, GO is expected to utilize as electrospun

fiber sheets because it provides advantages - ease of disper-

sion in polymer matrixes due to their oxygenated surface char-

acteristic, leading to stable dispersibility in aqueous-organic

solution by electrostatic repulsion.21,22)

In this study, we aimed to fabricate GO-decorated hybrid

fiber sheets composed of PLGA and col (GO-PLGA/Col) by

dual electrospinning and to characterize their physicochemical

properties for potential applications to tissue engineering

scaffolds.

Materials and Methods

Preparation of GO-PLGA/Col fiber sheetsPLGA resins (PLA/PGA = 75/25, MW = 70-110 kDa) were

purchased from Sigma-Aldrich (St. Louis, MO, USA). GO was

synthesized from expanded graphites according to a modified

Hummers and Offeman method.23) 20% (w/v, 200 mg/mL)

PLGA and 0.1% (w/w, 0.2 mg/mL) GO of the total PLGA

weight were dissolved in 1,1,1,3,3,3,-hexafluoro-2-propanol

(HFIP, Sigma-Aldrich, St. Louis, MO, USA) solvent to form the

first solution. The GO solution in HFIP was prepared by soni-

cating GO for at least 2 h to uniformly disperse GO particles.

Upon fabricating graphene sheets, the use of organic solvents

as media for the dispersion of graphene is poorly addressed

with high concentrations (> 1 mg/ml = 1000 ppm) of graphene,

whereas low concentrations (< 0.01 mg/ml = 10 ppm) of

graphene are well dispersed in organic solvents followed by

the mild sonication.24,25) 8.5% (w/v) Col (Darim Tissen, Seoul,

Korea) was dissolved in HFIP to form the second solution. As

the first solution, the GO-PLGA solution was placed in 5 mL

syringe fitted with a 25 G needle. A syringe pump (KD Scien-

tific, single-syringe infusion pump, Holliston, MA, USA) was

used to feed the GO-PLGA solution into the needle at the

flow rate of 0.5 mL/h. The 10 kV positive voltage by a high-

voltage power supply (NanoNC, Seoul, Korea) and a 11 cm

working distance between a needle tip and a collecting drum

were adopted for the electrospinning process. The second

solution containing Col was delivered to the 5 mL syringe fit-

ted with a 21 G needle and pushed to the needle tip at the

flow rate of 0.5 mL/h with another syringe pump. A voltage of

10 kV was applied and the working distance was 12 cm. The

GO-PLGA/Col hybrid fibers were collected on a rotating drum

wrapped with an aluminum foil. The rotating speed of the

grounded drum was 20 rpm. Collected GO-PLGA/Col hybrid

fiber sheets were subsequently vacuum-dried to remove any

residual solvents.

Physicochemical characterizations of of GO-PLGA/Colfiber sheets

The surface morphologies of GO-PLGA/Col hybrid fiber

sheets were observed under a field emission scanning electron

microscope (FESEM, Hitachi S-4700, Tokyo, Japan) at an acce-

lerating voltage of 5 kV, after platinum coating. The topogra-

phy of GO-PLGA/Col hybrid fiber sheets were characterized

by atomic force microscopy (AFM, non-contact mode, PSIA

XE-100, PSIA Inc., Fremont, CA, USA) with a Multi 75 silicon

scanning probe. Functional groups present in each sheet were

analyzed using a Fourier transform infrared (FTIR) spectros-

cope (FT/IR 6300, JASCO, Easton, MD, USA). The Raman

spectra of GO-PLGA/Col hybrid fiber sheets were obtained by

Macro Probe Raman Measurement System (Ramboss-500i,

Dongwoo Optron Co., Ltd, Kwangju-si, Korea) equipped with

a TE cooled CCD detector. The thermal stability of GO-PLGA/

Col hybrid fiber sheets was evaluated using a thermogravimetry

analyzer (TGA, TGA N-1000, Scinco, Seoul, Korea). The

heating rate was 10oC/min over the temperature range, room

temperature and 500oC. To determine the hydrophilicity of

GO-PLGA/Col hybrid fiber sheets, water contact angles were

measured by a contact angle goniometer (EasyDrop, model

FM40Mk2, Krüss, Hamburg, Germany) and then calculated

with dropshape analysis program.

Cell attachment and proliferation assaysHuman dermal fibroblasts (HDFs) from neonatal dermis

were kindly provided by Dr. Dong Kyun Rah (Department of

Plastic and Reconstructive Surgery, Yonsei University College of

Medicine, Seoul, Korea). HDFs were routinely maintained in

Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supple-

mented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis,

MO, USA) and a 1% antibiotic antimycotic solution (including

10,000 units penicillin, 10 mg streptomycin and 25 µg am-

photericin B per mL, Sigma-Aldrich, St. Louis, MO, USA) at

37oC in a humidified atmosphere of 5% CO2 in air. Studies

were performed with HDFs within 5 to 7 passages. PLGA,

GO-PLGA and GO-PLGA/Col hybrid fiber sheets were cut

into 10 × 10 mm2 and placed in a 24-well plate. All sheets

were sterilized under ultraviolet light overnight, followed by

washing with PBS containing a 2% antibiotic antimycotic solu-

20 Eun Ji Lee et al.

Biomaterials Research 2014

tion to remove any residual solvent and impurity. Cell

attachment and proliferation were determined by using a cell

counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan), which

contains highly water-soluble tetrazolium salt [WST-8, 2-(2-

methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-

2H-tetrazolium, monosodium salt], reduced to a water-soluble

formazan dye by dehydrogenases in cells. Typically, HDFs

were seeded at a density of 1 × 104 cells/mL on PLGA, GO-

PLGA and GO-PLGA/Col fiber sheets. According to the manu-

facturer’s instruction, each cell culture was incubated with

WST-8 solution in the last 4 hours of culture periods for cell

attachment (6 hours) or proliferation (1, 3 and 5 days) at 37oC

in the dark. Parallel sets of the cells cultured onto tissue cul-

ture plastics (TCP) were regarded as positive (+) controls. The

absorbance was determined at 450 nm in an ELISA reader

(SpectraMax 340, Molecular Device Co., Sunnyvale, CA, USA).

At the end of the incubation, the cellular morphology cultured

on GO-PLGA/Col hybrid fiber sheets was observed using AFM

as described above. In addition, proliferated cell morphologies

were observed by SEM as follows: Briefly, cultured cells were

washed with 0.1 M cacodylate buffer (pH 7.4) to remove

unattached cells. Cells were fixed with 2.5% glutaraldehyde

solution overnight at 4oC, dehydrated with a series of in-

creasing concentration of ethanol solution, and then vacuum-

dried. Fixed cell cultures were coated with an ultra-thin layer

of gold/platinum by an ion sputter (E1010, Hitachi, Tokyo,

Japan and then observed under a SEM (Hitachi S-800, Tokyo,

Japan) at an accelerating voltage of 20 kV.

Statistical analysisAll variables were tested in three independent cultures for

each experiment in vitro, which was repeated twice (n = 6).

Quantitative data are expressed as the mean ± standard de-

viation (SD). Data were tested for homogeneity of variances

using the test of Levene, prior to statistical analysis. Multiple

comparisons to detect the cellular behaviors of HDFs on GO-

PLGA/Col hybrid fiber sheets were carried out using one-way

analysis of variance (ANOVA) (StatView, SAS Institute, Cary,

NC, USA), which was followed by the Bonferroni test when

variances were homogeneous and the Tamhane test when

variances were not. A value of p < 0.05 was considered sta-

tistically significant.

Results and Discussion

The surface morphologic imaging by FESEM of pure PLGA,

GO-PLGA and GO-PLGA/Col hybrid fiber sheets are shown in

Figure 1(A). GO-PLGA and GO-PLGA/Col hybrid fiber sheets

were randomly oriented and have a three-dimensional inter-

connected pore structure which is similar to that of pure

PLGA fiber sheets. From FESEM images combined with image

analysis software, the diameter of GO-PLGA and GO-PLGA/

Col hybrid fibers was found to range between 250 nm and

890 nm (average fiber diameter: 480 ± 210 nm, 680 ± 185

nm respectively). There were no significant difference in the

morphologies according to existence of GO and Col.

Topographic imaging by AFM of pure PLGA, GO-PLGA and

GO-PLGA/Col hybrid fiber sheets are shown in Figure 1(B).

Figure 1. Surface morphological and topographical images of PLGA, GO-PLGA and GO-PLGA/Col hybrid fiber sheets: (A) FESEMimages and (B) 3D rendered images by AFM.


Vol. 18, No. 1

For each sample, a scan size of 30 × 30 µm was measured to

obtain a sufficient amount of ridges to characterize the

surfaces and to maintain enough resolution to achieve a

precise test. The structures found with the AFM measurement

correlate well with the FESEM images. The average roughness

of pure PLGA, GO-PLGA, GO-PLGA/Col hybrid fiber sheets

are 0.847 µm, 0.211 µm and 0.309 µm respectively. It was

recognized that the diameter of fibers have increasing ten-

dency, the roughness of fibers is also increased.

The Figure 2(A) shows the FT-IR spectra of Col, pure PLGA

fibers and GO-PLGA/Col hybrid fiber sheets. The spectra of

pure PLGA fibers showed that the C=O stretch and the C-O

stretch observed at around 1760 and 1080 cm−1, respectively.

Both bands exhibit carbonyl stretching demonstrating the pre-

sence of the ester group. The spectra of Col displayed mainly

bands at 1650, 1560, and 1230 cm−1, characteristic of the

amide I, II and III bands, respectively. The amide I absorption

is generated predominatingly by protein amide C=O stret-

ching vibrations and amide II is consisted of amide N-H ben-

ding vibrations and C-N stretching vibrations. The amide III

band is complex, consisting of components form C-N stret-

ching and N-H in plane bending from amide linkage.26) Addi-

tionally, bands at around 3550 and 2950 cm−1 were observed,

which represent the stretching of −OH and −CH3, respecti-

vely. By comparing the spectra of GO/PLGA-Col hybride fiber

sheets, it has been found that there are no additional peaks

excluding the specific bands of Col and PLGA. Raman spectra

of GO-PLGA and GO-PLGA/Col hybrid fiber sheets were

dominated by the noticeable band near 1350 cm−1 and

1600 cm−1, which was due to the D band and G band of GO

[Figure 2(B)]. The D band is activated once when the defects

are entered into sp2 hybridized carbon networks, and the

intensity of D band is proportional to the defects con-

centration.27,28) The G band is arising from the E2g vibrational

mode of sp2-carbon network. It is known fact that the

existence of strong D band at 1350□cm−1 must accompany

the existence of another defects-assisted Raman band, the D’

band, located at 1620□cm−1. The intensity of D’ band is also

proportional to the concentration of defects like as the case of

D band. And when the amount of defects was fairly in-

creased, the broadening effect of Raman bands was occurred.

Thus the broad G band of GO is usually considered as

consisting of both G and D’ bands despite the fact that it

looks like a single band due to the merge effect after

broadening.29) The specific bands of PLGA were also notified

in Raman spectrum. The noticeable band near 1768 cm−1 was

regarded as the ester group of PLGA. Also, the deformation

bands of CH2 and CH3 were observed at 1450 cm−1 and

1424 cm−1, respectively, which correspond to the anti-sym-

metric vibration of CH2 from the lactic unit of PLGA and from

the glycolic unit of PLGA.30) These results indicate that GO

was well dispersed in the surface of GO-PLGA and GO-PLGA/

Col hybrid fiber sheets. The Figure 2(C) shows the results of

TGA analysis. The main decomposition temperature was

measured as 395, 393 and 372oC for pure PLGA, for GO-

PLGA and for GO-PLGA/Col hybrid fiber sheets, respectively.

Each point of decomposition temperature at around 400oC is

signified the typical thermal degradation profile of PLGA.31)

The total mass loss of the GO-PLGA/Col was 89.97% which

was lower than 93.74% and 94.54% of pure PLGA and GO-

PLGA. This is due to the fact that the amount of PLGA was

decreased by adding the Col.32) Additionally, since the

Figure 2. Physicochemical and thermal properties of PLGA,GO-PLGA and GO-PLGA/Col fiber sheets: (A) FT-IR spectra, (B)Raman spectra and (C) TGA profiles.



graphene oxide is a thermally unstable materials, there was

slight mass loss at around 100oC in case of GO-PLGA and

GO-PLGA/Col hybrid fiber sheets. The mass loss in the early

stage means the pyrolysis of labile oxygen-containing groups of

graphene oxide.33) From TGA results, we recognized that GO,

PLGA and Col were well constructed in each sheet and GO-

PLGA/Col hybrid fiber sheet was thermally stable.

The hydrophilic/hydrophobic properties of electrospun

nanofiber sheets play essential roles in overall tissue engineer-

ing applications. As shown in Figure 3, the contact angles of

pure PLGA, GO-PLGA and GO-PLGA/Col hybrid fiber sheets

were 143.5 ± 4.8o, 127.9 ± 3.7o and 54.6 ± 3.7o, respectively.

Due to loading of GO which has abundant hydroxyl groups,

the contact angle of GO-PLGA sheets was significantly

(p < 0.05) decreased while its hydrophilicity of sheets was in-

creased. In addition, the contact angle of GO-PLGA/Col

hybrid fiber sheet was considerably decreased because of the

existence of Col. In general, contact angles are larger for

hydrophobic polymers with low surface energy than for more

hydrophilic materials with higher energy, which show a ten-

dency of higher cellular adhesion.34) It is suggested that GO-

PLGA and GO-PLGA/Col hybrid fiber sheets can better

enhance cell adhesion as they are more hydrophilic and have

higher surface energy than pure PLGA fiber sheets.

It is demonstrated that the GO-PLGA and GO-PLGA/Col

fiber sheets significantly (p < 0.05) improve initial attachment

of HDFs on the sheets as shown in Figure 4(A). The

uncertainty of GO biocompatibility was often a concern when

GO was used for biomedical applications. Many studies have

reported that the cytotoxicity of GO is dose-dependent and a

small quantity of GO does not exert adverse affects to cellular

behaviors.35-38) The improved hydrophilicity due to the hydro-

philic groups of GO provides higher HDFs adhesion than pure

PLGA. Furthermore, adding the Col into GO-PLGA sheets has

more favorable effects on cellular adhesion, since cells adhere,

spread and grow more easily on moderately hydrophilic

surfaces than on hydrophobic ones.39) It is considered that

relatively higher hydrophilicity and surface energy of GO-

PLGA/Col hybrid fiber sheets promote the adhesion of HDFs.

On the other hand, the proliferation pattern of HDFs on GO-

PLGA/Col fiber sheets showed the gradual increase during the

culture period, which was very similar to the TCP control

during the culture period [Figure 4(B)]. However, there was no

apparent difference in the proliferation patterns between

PLGA and GO-PLGA fiber sheets. This phenomenon can be

partly explained by the fact that GO has been shown to

Figure 3. Contact angles of PLGA, GO-PLGA and GO-PLGA/Col fiber sheets. Data are expressed as mean ± SD based on atleast duplicate observations from three independent experi-ments. Different letters denote significant differences betweenPLGA and the other sheets, p < 0.05. All photographs shownin this figure are representative of six independent experimentswith similar results.

Figure 4. Cellular behaviors on of HDFs on PLGA, GO-PLGAand/or GO-PLGA/Col fiber sheets. Cell attachment (A) and pro-liferation (B) were determined by a CCK-8 assay, and cellmorphologies (C) cultured on GO-PLGA/Col hybrid fiber sheetsfor 5 days were observed by AFM and SEM (magnification,× 500 and × 5,000 for insertion). Data are expressed as mean ±

SD based on at least duplicate observations from three inde-pendent experiments. Different letters denote significant differ-ences between TCP controls and cells cultured on fiber sheets,p < 0.05. All photographs shown in this figure are represen-tative of six independent experiments with similar results.


Vol. 18, No. 1

enhance growth and differentiation of mesenchymal, neural

and induced pluripotent stem cells.40-43) Moreover, Col

existence allows the biochemical interaction with cells which

mimics the integrin-ECM interaction during the culture

period.44) It thus seems that loading the GO and Col can play

a favorable role in the skin regeneration, while suitably affect

the proliferation and migration of dermal fibroblasts. This

pattern of cell proliferation on GO-PLGA/Col hybrid fiber sheets

was also partly evident from the morphological observations.

As shown in Figure 4(C), the cells cultured on GO-PLGA/Col

fiber sheets showed normal fibroblastic growth with a few

alterations in the cellular morphologies, which were almost

similar to those on PLGA/Col fiber sheets (data not shown).

Conclusion

Herein, we have demonstrated the feasibility of fabricating

GO-PLGA/Col hybrid fiber sheets using dual electrospinning.

Electrospun fiber sheets composed of various biodegradable

copolymers have been extensively examined in the context of

tissue engineering and regeneration; however, their use in

combination with GO is novel. GO-PLGA/Col hybrid fiber

sheets prepared in this study had a three-dimensional inter-

connected pore structure with diameter ranges from 250 nm

to 890 nm. The GO were uniformly dispersed in the polymer

matrix according to FT-IR and Raman results. The decomposi-

tion temperature of obtained fiber sheets decreases with the

addition of Col. GO and Col increased the hydrophilicity of

prepared fiber sheets. Taking these results into consideration, it

is suggested that GO-PLGA/Col hybrid fiber sheets have a

potential to support skin regeneration and can be exploited as

skin tissue engineering scaffolds.

Acknowledgement

This study was supported by Basic Science Research Pro-

gram through the National Research Foundation of Korea (NRF)

funded by the Ministry of Education, Science and Technology

(2012R1A2A2A02010181).

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