Available online at www.sciencedirect.com

Biomaterials 29 (2008) 2388e2399www.elsevier.com/locate/biomaterials

The immobilization of basic fibroblast growth factor on plasma-treatedpoly(lactide-co-glycolide)

Hong Shen, Xixue Hu, Jianzhong Bei, Shenguo Wang*

BNLMS, State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China

Received 10 November 2007; accepted 9 February 2008

Available online 7 March 2008

Abstract

In this study, possibility of the method of immobilization of basic fibroblast growth factor (bFGF) on polylactone-type polymer scaffolds viaplasma treatment was investigated. To introduce acid carboxylic functional groups on the surface of the polymer matrix, poly(lactide-co-glyco-lide) (PLGA) film was treated with carbon dioxide (CO2) plasma and then incubated in a phosphate buffer saline (PBS, pH 7.4) solution ofbFGF. The bFGF binding efficiency to the CO2 plasma-treated PLGA (PT-PLGA) films under different treating parameters was investigatedand compared. It was found bFGF binding efficiency to PLGA was enhanced by CO2 plasma treatment. The binding efficiency of bFGF toPLGA was variational with CO2 plasma treating time and it reached a maximum after a treating time of 20 min under the power of 20 W.The changes of surface chemistry and surface topography induced by CO2 plasma treatment played main roles in improving binding efficiency.Bound bFGF was released continuously from the films for up to 7 days in vitro. The stability of bFGF immobilized on PLGA film via CO2

plasma treatment was tested further under dynamic conditions by a Parallel Plate Flow Chamber. Mouse 3T3 fibroblasts were cultured onthe bFGF bound PLGA with a prior plasma treatment (20 W, 20 min) (PT-PLGA/bFGF) film, which showed that bFGF released from PT-PLGA/bFGF film was bioactive. Adhesion and growth of cells on PLGA scaffolds were greatly improved by immobilization of bFGF onthem. Therefore, the method of CO2 plasma treatment combining bFGF anchorage not only was usable in delivering bFGF, but also couldbe applied extensively for surface modification of scaffolds in tissue engineering.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Basic fibroblast growth factor (bFGF); Deliver vehicle; PLGA scaffold; CO2 plasma treatment; Surface modification; Tissue engineering

1. Introduction

Basic fibroblast growth factor (bFGF) is a member of a largefamily of structurally related protein that affects the growth,differentiation, migration and survival of a wide variety ofcell types [1,2]. It is a potent angiogenic factor in vivo andin vitro, and is mitogenic and chemotactic for both fibroblastand endothelial cells [3e7]. Administration of exogenousbFGF has shown therapeutic potential for tissue regeneration,wound healing, and angiogenesis [8e11]. However, the bioac-tivity of bFGF cannot always be expected when it is injectedinto the body by soluble form, because of its short durationof retention at wound sites and short half-life caused by

* Corresponding author. Tel./fax: þ86 10 62 581 241.

E-mail address: wangsg@iccas.ac.cn (S. Wang).

0142-9612/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biomaterials.2008.02.008

susceptibility to enzymatic and thermal degradation in vivo[12e14]. So, an effective delivery system of bFGF is requiredin order to overcome the shortcoming of bFGF therapy ina clinical setting.

Recently, a number of strategies and delivery vehicles havebeen designed and evaluated for the delivery of bFGF. Thesedelivery systems have been designed in a variety of geometriesand configurations, including two-dimension or three-dimen-sion scaffold, microsphere, and gel, and have been fabricatedfrom diverse types of natural and synthetic materials, includ-ing gelatin, collagen, hyaluronic acid, fibrin, heparin or hepa-rin-conjugated natural polysaccharides, polylactone-typebiodegradable polymers, and polyethylene glycol-based poly-mers [15e18]. bFGF is immobilized on the various shaped de-livery vehicles by either physisorption, ionic interaction orcovalent binding [19e21]. Although these strategies and deliv-ery vehicles have been considered to effectively deliver growth

2389H. Shen et al. / Biomaterials 29 (2008) 2388e2399

factors to some extent, an ideal method for their administrationremains unclear. Since polylactone-type biodegradable poly-mers, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA) and polycaprolactone (PCL),have been extensively used as scaffold materials for tissue en-gineering due to their good mechanical property, low immuno-genicity, non-toxicity, and adjustable degradation rate [22],research of the polymer scaffolds as carriers of bFGF hasmore important significance. The challenge lies in how to in-corporate the water-soluble growth factors into the hydropho-bic polymer scaffolds evenly. Growth factor can beincorporated directly into the polymer scaffolds at [23,24] orafter fabrication [25,26]. However, use of organic solvent dur-ing the polymer scaffolds fabrication process will damage bio-activity of bFGF. On the other hand, the poor hydrophilicityand lack of functional group of the polymers often results inlow loading efficiency of bFGF by solution dipping method af-ter fabrication. To improve ability of polylactone-type poly-mers binding bFGF, surface modification of the polymershas been developed [27,28]. However, since the functionalgroups are absent in the backbone of the polymers, it is diffi-cult to modify surface property by common chemical method.

Plasma treatment is an effective method for modifying sur-face property of a material such as wettability, topography,surface charge states and biocompatibility, while it has littleeffect on bulk properties of the material [29e31]. Plasmatreatment is also a rapid, clear and non-solvent surface modi-fication method that can be used to introduce some specific el-ement or functional group onto the surface of a polymer onlyby selecting and applying suitable gas. These specific func-tional groups provide special chemical reactivity and varyingphysical properties for surface. The variability of surfacewas essential for functionalization with additive molecules.So, by plasma treatment method a variety of molecules, suchas protein, peptide and gelatin, have been successfully boundon surface of some synthetic polymer matrices [32e35].

Considering bFGF is also protein, it is significant and inter-esting whether the bFGF can be bound efficiently on polylac-tone-type polymer matrices by plasma treatment, as well aswhether the bound bFGF can keep its bioactivity and sustainrelease from the polymer matrix. It has been reported thatmore pC]O or eCOOH can be incorporated into surfaceof the polymer by carbon dioxide plasma treatment [36]. Sincecarboxylic groups can easily desorb proton, there may bestrong electrostatic interaction between CO2 plasma-treatedpolymer and bFGF that is a positively charged protein at phys-iological pH (pI¼ 10.0). In the present study, CO2 plasmatreatment was chosen to provide negative charged groups forthe surface of PLGA matrices and then bFGF was bound onthe plasma-treated PLGA matrices. Following that effect ofCO2 plasma treatment on binding bFGF was tested by releaseof the bound bFGF from the plasma-treated PLGA film in vi-tro. Moreover, the stability of bFGF bound to PLGA film wasfurther tested under shear stress by a Parallel Plate FlowChamber [37,38]. The bioactivity of released bFGF was eval-uated by measuring ability of the released bFGF to stimulateproliferation of 3T3 fibroblasts. Finally, the influence of

immobilized bFGF on adhesion and growth of cells inPLGA scaffold was investigated.

2. Materials and methods

2.1. Materials

PLGA (molar ratio of lactyl/glycotyl¼ 70/30, Mw¼ 110,000) was pre-

pared by ring-opening polymerization of L-lactide (PURAC, Netherlands) and

glycolide (PURAC, Netherlands) under high vacuum at 160 �C for 20 h in the

presence of stannous octoate (SIGMA, German) as catalyst (0.05 wt%) [39].

2.2. Preparation of PLGA film and scaffold

The film with dense structure was prepared by casting 5 wt% PLGA chlo-

roform solution into a poly(tetrafluoroethylene) (PTFE) mould. After solvent

evaporation in air at room temperature, the formed film was removed from

the mould and dried in vacuum at room temperature for 48 h. The thickness

of PLGA film was 0.1 mm, which was determined by micrometer.

PLGA scaffold with porous structure was manufactured by an improved sol-

ideliquid phase separation method [40]. A certain weight of sieved NaCl gran-

ules (diameter 200e280 mm) was added into 5% (w/v) solution of PLGA in

dioxane, then the slurry was maintained at 0 �C for over 24 h to induce solideliq-

uid phase separation completely. After the solvent was removed by freeze-drying

for 3 days, the formed matrix was put into distilled water to leach the NaCl out.

Finally, the PLGA scaffold was dried and kept in a desiccator for usage.

2.3. Plasma treatment of PLGA film and scaffold

Plasma treatment was carried out on Samco Plasma Deposition (Model

PD-2, 13.56 MHz) under CO2 atmosphere. PLGA film or scaffold was placed

on the electrode in the plasma chamber. The chamber was evacuated to less

than 10 Pa before filling with CO2 gas. After pressure of the chamber was sta-

bilized to 20 Pa, glow discharge plasma was created by controlling electrical

power at 20 W and radio frequency of 13.56 MHz for a predetermined time.

Finally, the plasma-treated sample was further exposed to CO2 atmosphere

for another 10 min before the sample was taken out from the chamber.

2.4. Characterization of PLGA film

Surface chemical compositions of untreated and plasma-treated PLGA

films were investigated by X-ray photoelectron spectrometer (XPS) (ESCA-

Lab220i-XL, VG Scientific), and concentration of various C1s speaks were

calculated from the relative C1s peak area. The topography of various

PLGA films was examined by atomic force microscopy (AFM) (Digital instru-

ments Inc., Santa Barbara, CA). Three-dimensional images and surface topog-

raphy parameters (Ra, PeP and PeV) data were acquired using Nanoscope

image-processing software. Contact angle of various PLGA films to deionized

water was measured on air surface of the samples using a FACE CA-D-type

Contact Angle Meter (Kyowa Kaimenka-gaku Co., Ltd). Ten independent de-

terminations at different sites of a sample surface were averaged.

2.5. bFGF binding study

Untreated and plasma-treated (20 W, 5, 10, 20 and 30 min) PLGA films

with a diameter of 10 mm were placed the bottom of a 48-well plate. Solution

of 500 ng/ml bFGF (human recombinant, 154 amino acid, Mw¼ 17.2 kDa,

PeproTechAsia) in phosphate buffer saline (PBS, pH 7.4) was prepared and

200 ml bFGF solution was added to each well of the 48-well plate. After the

PLGA films were incubated in the bFGF solution for 1 h at room temperature

on a shaker, supernatants were collected, respectively. Then bFGF bound films

were washed with PBS for two times. All the washings were also collected and

mixed with previous supernatants, respectively. The amount of bFGF in the

collected mixed solution was assayed using a Quankine Immunoassay kit ac-

cording to the manufacture’s instruction (Human bFGF Quankine ELISA kit,

R & D Systems, Minneapolis, MN, USA). Binding efficiency of bFGF to the

PLGA films was evaluated according to the following formula:

2390 H. Shen et al. / Biomaterials 29 (2008) 2388e2399

Binding efficiency ð%Þ ¼ ½ðWa �WbÞ=Wa� � 100

where Wa and Wb are the weight of bFGF in PBS solution before and after in-

cubating PLGA films, respectively.

In the research, untreated PLGA and plasma-treated PLGAwere, respectively,

abbreviated as UT-PLGA and PT-PLGA. bFGF bound PLGA without a prior

plasma treatment and bFGF bound PLGA with a prior plasma treatment (20 W,

20 min), respectively, abbreviated as UT-PLGA/bFGF and PT-PLGA/bFGF.

2.6. bFGF release study

UT-PLGA/bFGF and PT-PLGA/bFGF films with a diameter of 10 mm

were immersed in 500 ml release medium, respectively, which was phosphate

buffer saline (PBS, pH 7.4) solution supplemented with 1% bovine serum al-

bumin (Sigma) to perform release study. The films were incubated at 37 �C up

to 3 weeks under static condition. At preset time intervals, the incubation so-

lution was collected and replaced with fresh release medium. The amount of

released bFGF in the collected medium was determined by a Quankine Immu-

noassay kit according to the manufacture’s instruction.

2.7. Stability of bound bFGF under dynamic conditions

Stability of bound bFGF was studied under shear stress field in a Parallel

Plate Flow Chamber. The detailed dimensions of the Parallel Plate Flow Cham-

ber and circuit flow system were referred in a previous work [37,38]. At first the

UT-PLGA/bFGF and PT-PLGA/bFGF films with a diameter of 10 mm were im-

mobilized on the lower glass plate of the flow chamber and the upper glass plate

was assembled in the flow chamber. Then PBS solution with predetermined

flowing rate was driven by a peristaltic pump and the temperature of PBS so-

lution was controlled at 37 �C. After the films had been exposed to a shear

stress of 11.5 N/m2 (PLGA example) for 10 min, the films were taken out

from the glass plate. Then bFGF bound on the UT-PLGA and PT-PLGA

(20 W, 20 min) films exposed to shear stress was assayed and compared.

2.8. Preparation of cells

Mouse 3T3 fibroblasts were supplied by the Chinese Academy of Military

Medical Sciences. The cells were incubated at 37 �C in DMEM (Gibco) sup-

plemented with 10% fetal bovine serum (Gibco) and 100 U/cm3 each of pen-

icillin and streptomycin in a 5% CO2 incubator. When the cells had grown to

confluence, they were detached by trypsin/EDTA (0.05%/0.02%) (Sigma) and

seeded onto various PLGA samples.

2.9. bFGF bioactivity assay

3T3 fibroblasts at a density of 1.5� 104 cells/well were evenly seeded onto 24-

well culture plate with addition of 1 ml of DMEM basal medium including 5% fe-

tal bovine serum. UT-PLGA, PT-PLGA (20 W, 20 min), UT-PLGA/bFGF and PT-

PLGA/bFGF films with a diameter of 15 mm were immersed in the DMEM basal

medium of the well, respectively, and cultured at 37 �C under 5% CO2. As a pos-

itive control, 3T3 fibroblasts were cultured in 1 ml DMEM basal medium includ-

ing 5% fetal bovine serum and fresh bFGF added daily at the same concentration as

that of the bFGF released from PT-PLGA/bFGF films. The culture medium was

changed daily. At predetermined interval, the cells on the wells were digested

by trypsin/EDTA and the number of cells in the wells was counted.

2.10. Cell adhesion study under dynamic condition

The interaction of various PLGA films with mouse 3T3 fibroblasts was

quantitatively evaluated using a modified Parallel Plate Flow Chamber accord-

ing to previous work [37,38]. Mouse 3T3 fibroblast suspension with a density

of 1.2e1.5� 105 cells/ml was immediately seeded on various PLGA films

(about 1.6� 104 cells/cm2) covered the lower glass plates of the chamber

and cultured in an incubator. After the 3T3 fibroblasts were cultured for 4 h

under static condition, the cell-seeded glass plate and the upper glass plate

were assembled in the flow chamber. An image was captured arbitrarily in

the center of flow chamber as the starting point by invert light microscopy

(Olympus Optical Co., Ltd). Then the flow of culture medium was initiated

by turning on the peristaltic pump to control a proper flow rate. The changes

of cell adhesion under shear stress were recorded by taking photos every 1 min

and the fraction of adherent cells with time under shear stress was obtained by

calculating the number of cells at different times using image analysis

software.

2.11. MTT assay

UT-PLGA and PT-PLGA (20 W, 20 min) scaffolds (10� 10� 4 mm3)

were immersed in 2 ml bFGF solution (500 ng/ml), respectively, for 1 h at

room temperature on a shaker. Then the scaffolds were washed with PBS

and subsequently water. After the scaffolds were dried by freeze-drying, the

UT-PLGA/bFGF and PT-PLGA/bFGF scaffolds were obtained.

Various PLGA scaffolds (10� 10� 4 mm3) were placed into a 24-well

culture plate and cell suspension (100 ml) with a cell density of

5� 105 cells/ml was seeded onto the scaffolds. The cell-seeded scaffolds

were maintained at 37 �C under 5% CO2 for 3 h, and then 1.5 ml of culture

medium was added to each well. At predetermined interval, 15 ml of MTT so-

lution (5 mg/ml in PBS) was added to each well, followed by incubation at

37 �C for 4 h to MTT formazan formation. Then upper medium was carefully

removed and the intracellular formazan was dissolved by adding 800 ml of

0.04 mol/l HCl/iso-propanol to each well. The absorbance of produced forma-

zan was measured at 570 nm with microplate reader (ZS-2, Beijing).

2.12. Observation of cell morphology

Various scaffolds (10� 10� 4 mm3) were located in a 24-well culture

plate. Cell suspension (8� 105 cells/ml) was seeded on the scaffolds until the

scaffolds became saturated and then they were cultured for 3 h prior to addition

of 1.5 ml culture medium into culture plate. After being cultured for 2 weeks,

the scaffolds were taken out from culture plate and washed with PBS, then fixed

with 2.5% glutaraldehyde for 24 h at 4 �C. After the scaffolds were dehydrated

through a series of graded alcohols and dried, they were sputter-coated with

gold. Finally cell morphology was observed by SEM (Hitachi S-530, Japan).

2.13. Statistical analysis

The data were expressed as means� standard deviations (SD) (n¼ 3 or 4).

Statistical comparisons were performed using the Student’s t-test. p-Val-

ues< 0.05 were considered statistically significant.

3. Results and discussion

3.1. Effect of CO2 plasma treatment on surfacetopography of PLGA film

The surface topography of PLGA films before and afterCO2 plasma treatment was investigated by AFM. The three-di-mensional images of AFM were shown in Fig. 1 and the quan-titative data of surface roughness parameters were presented inTable 1. The results showed that surface morphology of PLGAfilm exhibited drastic variations after CO2 plasma treatment.The surface of UT-PLGA was almost smooth (Fig. 1a) andthe value of arithmetic mean deviation of surface (Ra) wasonly 0.4 nm (Table 1). After a treating time of 5 min, the sam-ple surface became obviously rough (Fig. 1b). The roughnesswas increased to Ra¼ 6.6 nm, PeP¼ 125.0 nm, PeV¼ 30.3 nm, where PeP and PeV were maximum meanpeak-to-peak width of surface and maximum mean peak-to-valley height of surface, respectively. After a treating time

Fig. 1. AFM images of PLGA films before and after CO2 plasma treatment. (a) Untreated; (b) treated at 20 W for 5 min; (c) treated at 20 W for 10 min; (d) treated

at 20 W for 20 min; (e) treated at 20 W for 30 min.

2391H. Shen et al. / Biomaterials 29 (2008) 2388e2399

of 10 and 20 min, the film surface showed more obvious undu-late characteristics (Fig. 1c and d). Many large valleys andpeaks with nano-scale were produced and the surface rough-ness parameters of Ra, PeP, and PeV increased to 11.6,495.6, and 41.1 nm, respectively, after a treating time of20 min. With further increasing treating time to 30 min, themorphology variation was much more remarkable. Some val-leys and peaks with micron-scale (Fig. 1e) were produced andthe PLGA film with high surface parameters (Ra¼ 16.8 nm,PeP¼ 1522.0 nm, PeV¼ 86.7 nm) was obtained. In general,the etching effect of plasma treatment on polymer surface was

produced by degradation of the polymer surface during plasmatreatment processes. By adjusting plasma etching time, sur-faces of PLGA films with different roughness from nano- tomicron-scale could be obtained.

3.2. Effect of CO2 plasma treatment on surface chemistryof PLGA film

The surface chemical composition of PLGA films before andafter CO2 plasma treatment was determined by XPS measure-ment. The C1s peaks were resolved into three peaks with binding

Table 1

Surface roughness parameter of PLGA films before and after CO2 plasma

treatment

Surface

parameter

CO2 plasma treatment

None 20 W,

5 min

20 W,

10 min

20 W,

20 min

20 W,

30 min

Ra (nm)a 0.4 6.6 9.1 11.6 16.8

PeP (nm)b 1.3 125.0 237.5 495.6 1522.0

PeV (nm)c 0.8 30.3 36.3 41.1 86.7

a Arithmetic mean deviation of surface.b Maximum mean peak-to-peak width of surface.c Maximum mean peak-to-valley height of surface.

2392 H. Shen et al. / Biomaterials 29 (2008) 2388e2399

energies of 284.6, 286.4 and 288.6 eV, which were attributed tocarbons in eCeH or eCeCe, eCeOe, and pC]O or eCOOH groups, respectively (Fig. 2). The relative composition ofthe three types of C1s changed with time of plasma treatment un-der 20 Wof power, as shown in Table 2. The fraction of pC]O oreCOOH increased from 30.2% to 40.9% with treating time from0 to 20 min and further increasing treating time the fraction ofpC]O or eCOOH almost remained constant. While the fractionof eCeOe quickly increased to 44.9% after treatment for 5 minand then fell to 35.0% after 10 min treatment. After treating timelonger than 10 min, no big change of the fraction of eCeOecould be observed. The fraction ofeCeH or eCeCehad justa re-verse change to that of eCeOe. The above results could be ex-plained that after 5 min treatment, a part of eCeH or eCeCebonds were oxidized to eCeOe and pC]O or eCOOH bondsby generated active species, which led to an increase in the fractionof eCeOe and pC]O or eCOOH and a decrease in eCeH or eCeCe bonds. With prolonged treatment, the existing eCeH or eCeCe and eCeOe can be oxidized continuously and changedinto pC]O or eCOOH bonds, while eCeCe and eCeOecan be cleaved into new eCeH or eCeCe bonds. Therefore,the subsequent decrease in number of eCeOe after 5 min treat-ment may be ascribed to the faster oxidization and cleavage ratesof eCeOe than its formation rate. The results indicated thata maximal value of eCeOe or pC]O or eCOOH functionalgroups could be obtained by controlling plasma treating time.

3.3. Effect of CO2 plasma treatment on surfacehydrophilicity of PLGA film

Surface hydrophilicity of PLGA film was identified bymeasuring contact angle to water and the higher hydrophilicfilm has a smaller water contact angle. The contact angle ofPLGA film to water decreased with increasing CO2 plasmatreatment time. After a treating time of 5 min, the contact an-gles of PLGA film to water decreased from 78� to 43�, whichmeant that the hydrophilicity of PLGA film increased greatlyby CO2 plasma treatment. When the sample underwent treat-ment for 30 min, the water contact angles were less than20�. The increase of hydrophilic functional groups (eCeOeand pC]O or eCOOH) and roughness of surface were rea-sons for improvement of hydrophilicity of the PT-PLGA films.

3.4. Effect of CO2 plasma treatment on binding bFGF ofPLGA film

The binding efficiency of bFGF to UT-PLGA and PT-PLGA films was shown in Fig. 3. The result showed CO2

plasma treatment led to increase of bFGF binding efficiency.After a treating time of 5 min, the bFGF binding efficiencywas significantly increased from 16.1% to 33.5%. With pro-longing treating time, the bFGF binding efficiency graduallyincreased and a maximum value (66.3%) was observed forthe PLGA film treated for 20 min, which was over four timeshigher than that of the UT-PLGA film. However, when plasmatreating time was further increased to 30 min, the binding ef-ficiency of bFGF to PLGA film exhibited decrease.

The enhanced binding efficiency by CO2 plasma treatmentcould be explained from two aspects e surface chemistry andsurface topography. In this study, the XPS data (Table 2) showedthat controlling CO2 plasma treating time could enhance thenumber of eCeOe and pC]O or eCOOH (electron-endow-ing groups) on PLGA surface and its negative charge densitycould be presumed to increase. Since bFGF is a positivelycharged protein at physiological pH (pI¼ 10.0), the enrichedpolar O-containing groups (eCeOe and pC]O or eCOOH)on the surface of PT-PLGA films could provide many sites tocatch bFGF by electrostatic interaction. The hydrogen bondingbetween these rich O-containing groups and bFGF also could beproduced, which further contributed to bFGF binding. In addi-tion, it can be seen that CO2 plasma treatment can producednano-structured surface topography on the PLGA film fromthe AFM results (Fig. 1 and Table 1). The nano-structured sur-face topography increased surface/volume ration, which couldprovide more binding sites for bFGF and thus enhanced thebinding efficiency. After 5 min of CO2 plasma treatment, the en-hanced binding stemmed from the synergetic effects of the polarO-containing groups and the rough topography surface. After10 min of treatment, although the eCeOe decreased comparedwith plasma treating 5 min, the acid eCOOH increased fast,which more easily desorbed proton. So O-containing groups(mainly came from eCOOH) and rougher surface made thebinding efficiency further enhance. With further increasingtreating time to 20 min, although the chemistry compositionof the PLGA film had no big change, the bFGF binding effi-ciency continuously increased because its surface was rougherthan that of the PLGA film treated for 10 min. The decrease ofbFGF binding to the PLGA film treated for 30 min might resultfrom high hydrophilicity and micro-structured surface. In gen-eral, too high hydrophilicity of materials is disadvantage to ad-sorb proteins on it [41]. Moreover, bFGF is a single chain proteincomposed of 154 amino acids, which might be preferentiallyadsorbed to the surface with nano-structured topography.

3.5. Release behavior of bFGF from CO2 plasma-treatedPLGA film

The release profiles of bFGF from UT-PLGA/bFGF andPT-PLGA/bFGF films were determined, as shown in Fig. 4.The UT-PLGA/bFGF film showed a rapid release at the first

Table 2

Fraction of carbon functional groups from high-resolution C1s XPS peaks of

PLGA films before and after CO2 plasma treatment

CO2 plasma

treatment

288.6 eV pC]O

or eCOOH (%)

286.4 eV

eCeOe (%)

284.6 eV eCeH or

eCeCe (%)

None 30.2 32.9 36.9

20 W, 5 min 32.3 44.9 22.8

20 W, 10 min 39.9 35.0 25.1

20 W, 20 min 40.9 34.7 24.4

20 W, 30 min 41.0 34.0 25.0

294 292 290 288 286 284 282 280

>C=O-C-O-

-C-C-

Binding energy (eV)

294 292 290 288 286 284 282 280

-C-O--C-C->C=O

Binding energy (eV)

294 292 290 288 286 284 282 280

>C=O -C-O-

-C-C-

Binding energy (eV)

294 292 290 288 286 284 282 280

-C-C-

-C-O->C=O

Binding energy (ev)

294 292 290 288 286 284 282 280

-C-C-

-C-O->C=O

Binding energy (eV)

a b

c d

e

Fig. 2. High-resolution XPS spectra of C1s region of PLGA films before and after CO2 plasma treatment. (a) Untreated; (b) treated at 20 W for 5 min; (c) treated at

20 W for 10 min; (d) treated at 20 W for 20 min; (e) treated at 20 W for 30 min.

2393H. Shen et al. / Biomaterials 29 (2008) 2388e2399

day followed by a very slow release. However, the PT-PLGA/bFGF film exhibited a continuous release pattern about 7 daysafter a moderate burst release. The different bFGF release pro-files of the two films could be attributed to the strong electro-static interaction and hydrogen bonding between bFGF andPT-PLGA films. Since the polar O-containing groups on UT-PLGA film were less, most bFGF was adsorbed by a weak in-teraction, which resulted in the rapid desorption of bFGF uponcontacting with aqueous medium. However, on PT-PLGA(20 W, 20 min) film some bFGFePLGA complexes could beformed by strong electrostatic interaction and hydrogen

0

10

20

30

40

50

60

70

80

30201050

*#

**

*

bF

GF

b

in

din

g e

ffic

ie

nc

y (%

)

CO2 plasma treating time (min)

Fig. 3. Binding efficiency of bFGF to PLGA films before and after CO2 plasma

treatment. *p< 0.05; significant against the binding efficiency of bFGF to UT-

PLGA film; #p< 0.05, significant against the binding efficiency of bFGF to

PT-PLGA (20 W, 5, 10 and 30 min) films. Circular UT-PLGA and PT-

PLGA (20 W, 5, 10, 20 and 30 min) films with a diameter of 10 mm were in-

cubated for 1 h with 200 ml bFGF solution (500 ng/ml).

0 1 2 3 4 5 6 7 8 9-1

0

1

2

3

4

5

6

7

8

Cu

mu

la

tiv

e re

le

as

e o

f b

FG

F (n

g / film

)

Time (days)

UT-PLGA/bFGFPT-PLGA/bFGF

Fig. 5. Cumulative releases of bFGF from UT-PLGA/bFGF and PT-PLGA/

bFGF films with a diameter of 10 mm after exposed to a shear stress of

11.5 N/m2 for 10 min. bFGF was not detected in the release medium from

UT-PLGA/bFGF film after exposed to a shear stress of 11.5 N/m2 for

10 min. Circular UT-PLGA and PT-PLGA (20 W, 20 min) films with a diame-

ter of 10 mm were loaded for 1 h with 200 ml bFGF solution (500 ng/ml).

25 UT-PLGAPT-PLGA(20W, 20 min)

2394 H. Shen et al. / Biomaterials 29 (2008) 2388e2399

bonding. Therefore, bFGF in the complexes was presumablyreleased out in a more sustained manner. It was reported thatthe growth factor was released from carrier mainly by a diffu-sion-controlled mechanism, an erosion mechanism or a combi-nation with diffusion and a thermodynamic equilibriummechanism between free bFGF in the release medium andbFGF bound to carrier [17,42]. Since most bFGF was weaklybound on the surface of UT-PLGA, the bFGF release from UT-PLGA could mainly be a quick diffusion process. However,the bFGF release from PT-PLGA (20 W, 20 min) might in-volve two distinct release mechanisms. The initial burst re-lease from PT-PLGA was caused mainly by a quickdiffusion of weakly bound bFGF and the subsequent sustained

0 5 10 15 20 250

5

10

15

20

25

Cu

mu

lative release o

f b

FG

F (n

g / film

)

Time (days)

UT-PLGA/bFGFPT-PLGA/bFGF

Fig. 4. Cumulative releases of bFGF from UT-PLGA/bFGF and PT-PLGA/

bFGF films with a diameter of 10 mm. Circular UT-PLGA and PT-PLGA

(20 W, 20 min) films with a diameter of 10 mm were loaded for 1 h with

200 ml bFGF solution (500 ng/ml).

bFGF release was controlled by a thermodynamic equilibriumbetween the bFGFePLGA complexes and free bFGF in re-lease medium. Hence, the CO2 plasma treatment combiningwith bFGF anchorage was an effective method in controllingsustained bFGF release.

3.6. Stability of bound bFGF under dynamic conditions

To simulate the situation inside body, it is necessary to ex-tend the in vitro study of materialebFGF interaction to shear

0 2 4 6 80

5

10

15

20

*

*

*

*

Cell n

um

ber (*10

4)

Time (days)

UT-PLGA/bFGFPT-PLGA/bFGFPositive control

Fig. 6. Proliferation of 3T3 fibroblasts cultured in the basal medium containing

various PLGA films with a diameter of 15 mm within different period. Positive

control: proliferation of 3T3 fibroblasts cultured in basal medium with daily

additions of bFGF in a free form at the same concentration as that of the

bFGF released from PT-PLGA/bFGF films; *p> 0.05, insignificant against

positive control. Circular UT-PLGA and PT-PLGA (20 W, 20 min) films

with a diameter of 15 mm were loaded for 1 h with 400 ml bFGF solution

(500 ng/ml).

Fig. 7. Light micrographs of cells cultured on various PLGA films under different condition. The film from top line to bottom line was: top line (aec) UT-PLGA;

second line (def) PT-PLGA (20 W, 20 min); third line (gei) UT-PLGA/bFGF; bottom line (jel) PT-PLGA/bFGF. The cells culture condition from left rank to right

rank was: left rank (a, d, g and j) cells cultured under no shear stress for 4 h; middle rank (b, e, h and k) after 4 h of static culture, the cells further cultured under

36.5 N/m2 of shear stress for 10 min; right rank (c, f, i and l) after 4 h of static culture, the cells further cultured under 36.5 N/m2 of shear stress for 60 min.

2395H. Shen et al. / Biomaterials 29 (2008) 2388e2399

stress field. Since bFGF bound on the PLGA films couldn’t bemeasured directly, to understand the amount of bFGF re-mained on films exposed to the shear stress, the release ofbFGF from UT-PLGA/bFGF and PT-PLGA/bFGF films ex-posed to a shear stress of 11.5 N/m2 (PLGA example) for10 min was investigated and compared. Results showedbFGF was not detected in the release medium from the UT-PLGA/bFGF film exposed to shear stress, while the releaseof bFGF from PT-PLGA/bFGF film exposed to shear stresswas obvious and exhibited a continuous release pattern about7 days, as shown in Fig. 5. Since most bFGF was adsorbed on

the UT-PLGA film by a weak interaction due to lack of the po-lar O-containing groups on it, little bFGF remained on the UT-PLGA film exposed to a shear stress. However, most of bFGFbound on the PT-PLGA (20 W, 20 min) film by strong electro-static interaction and hydrogen bonding was enough to resistthe shear stress to some extent and thus a lot of bFGF couldremain on the PT-PLGA (20 W, 20 min) film when the PT-PLGA/bFGF film had exposed to shear stress for a longertime. Therefore, the stability of bFGF bound to the PLGAfilm under dynamic conditions was greatly improved by CO2

plasma treatment combining with bFGF anchorage.

2396 H. Shen et al. / Biomaterials 29 (2008) 2388e2399

3.7. Bioactivity assay of released bFGF

0 10 20 30 40 50 600

20

40

60

80

100

UT-PLGAPT-PLGA (20 W, 20 min)UT-PLGA/bFGFPT-PLGA/bFGF

Pe

rc

en

ta

ge

o

f a

dh

ere

nt c

ells

(%

)

Time (min)

Fig. 8. The fraction of 3T3 fibroblasts adhered on various PLGA films as the

function of time under 36.5 N/m2 of shear stress.

The biological activity of bFGF released from PLGA filmswas evaluated by measuring its ability to stimulate proliferationof 3T3 fibroblasts. As shown in Fig. 6, 3T3 fibroblasts in thebasal medium containing UT-PLGA or PT-PLGA (20 W,20 min) film showed low proliferation. Although 3T3 fibro-blasts in the basal medium containing UT-PLGA/bFGF filmexhibited modest proliferation in the first 2 days, proliferationwas obviously lower than that in the basal medium containingPT-PLGA/bFGF film thereafter. This was likely due to a rapidrelease of bFGF from the UT-PLGA film at the first day anda very slow release in following days. However, the prolifera-tion of 3T3 fibroblasts in the basal medium containing the PT-PLGA/bFGF film was rapid, and the cell growth for 8 days inthe basal medium containing the PT-PLGA/bFGF film was notany statistically different from that in the basal medium con-taining free bFGF added daily. The results meant sustained re-lease of bFGF from the PT-PLGA/bFGF film preserved itsbioactivity, which was almost same as that of fresh bFGF dailyadded to the basal medium in a free form. So, the modifiedPLGA with bFGF could have therapeutic potential for tissue re-generation, wound healing, and angiogenesis.

3.8. Effect of immobilization of bFGF on cell adhesion

0.0

0.1

0.2

0.3

0.4

0.5

0.6

1074

*#*

*#$*#$

*#$***

UT-PLGAPT-PLGA (20W, 20 min)UT-PLGA/bFGFPT-PLGA/bFGF

Ab

so

rb

an

ce

Culture time (days)

Fig. 9. MTT assay of 3T3 fibroblasts on various PLGA scaffolds within differ-

ent period; *p< 0.05, significant against the proliferation and viability of 3T3

fibroblasts on the UT-PLGA scaffold at the corresponding day; #p< 0.05, sig-

nificant against proliferation and viability of 3T3 fibroblasts on the UT-PLGA/

bFGF scaffold at the corresponding day; $p< 0.05, significant against prolif-

eration and viability of 3T3 fibroblasts on the PT-PLGA (20 W, 20 min) scaf-

fold at the corresponding day.

In order to simulate the situation inside the body, the mate-rialecells interaction was studied under shear stress field. TheParallel Plate Flow Chamber has been proven to be a suitabledevice to study cell adhesion on surface of materials undera shear stress field [37,38,43,44]. Fig. 7 showed the light mi-crographs of 3T3 fibroblasts adhered to various PLGA filmsunder different condition. It could be seen that after being cul-tured for 4 h under static condition, most cells adhered to UT-PLGA film were round and only few cells spread (Fig. 7a).Moreover, most cells were removed from the UT-PLGA filmswithin short time under shear stress (Fig. 7b and c). It was ob-vious the cells on the PT-PLGA (20 W, 20 min) under staticculture spread better than on the UT-PLGA and many cellsshowed a flat shape (Fig. 7d). Under shear stress the cells onthe PT-PLGA (20 W, 20 min) gradually withdrew from theirborders and with increasing time under shear stress, cell extru-sion was retracted to the cell body and then the cell was de-tached from the UT-PLGA film (Fig. 7e and f). The resultmeant the ability of cells on PLGA film to endure shear stresswas greatly improved after modified by CO2 plasma treatment.At the same time, the cells on the UT-PLGA/bFGF understatic culture showed spherical and spread morphology(Fig. 7g) and most cells were removed from the UT-PLGA/bFGF (Fig. 7h and i) film exposed to shear stress. However,many cells on the PT-PLGA/bFGF films under static cultureshowed a flat shape and protruded many filopodia that tightlyanchored to the substrate (Fig. 7j). When the cells on the PT-PLGA/bFGF gradually withdrew from their borders undera shear stress field, the pseudopods of cells hold back detach-ment of cells from the film (Fig. 7k and l). The result indicatedthat the ability of cells on PLGA film to endure shear stress

was further improved by immobilizing bFGF on the PT-PLGA.

The curve of percentage of adherent cells vs. time wasshown in Fig. 8. It could be clearly seen that the cells quicklydetached from the UT-PLGA film and about 76.0% cells haddetached within 10 min under shear stress. For UT-PLGA/bFGF film, the percentage of adherent cells declined to19.3% after 60 min under shear stress. However, the cellsgradually detached from PT-PLGA (20 W, 20 min) and PT-PLGA/bFGF films and percentage of adherent cells was76.7% and 95.6%, respectively, after 60 min under shearstress.

The results indicated PT-PLGA/bFGF was most favorablefor cell adhesion. Improved hydrophilicity, rough surface,rich O-containing groups and more binding bFGF of

Fig. 10. SEM images of cross section of PLGA scaffolds before and after 3T3 fibroblasts culture for 2 weeks: (a) macroscopic PLGA scaffold before cell culture

(100�); (b) microscopic PLGA scaffold before cell culture (600�); (c) UT-PLGA scaffold after cell culture (600�); (d) PT-PLGA (20 W, 20 min) scaffold after

cell culture (600�); (e) UT-PLGA/bFGF scaffold after cell culture (600�); (f) PT-PLGA/bFGF scaffold after cell culture (600�). The arrows pointed to the cells.

2397H. Shen et al. / Biomaterials 29 (2008) 2388e2399

PT-PLGA/bFGF film may play an important role in enhancingcell resistance to shear stress. Therefore, the quality of celladhesion of PLGA scaffold had been greatly improved byCO2 plasma treatment combining with bFGF anchorage.

3.9. Effect of immobilization of bFGF on cell growth

According to tissue engineering application, porous PLGAscaffolds were also fabricated and immobilized bFGF by CO2

plasma treatment combining with bFGF anchorage.

Proliferation and viability of 3T3 fibroblasts cultured onvarious PLGA scaffolds were determined by MTT assay aftercultured for 4, 7 and 10 days, as shown in Fig. 9. It could beseen that the proliferation of cells on the UT-PLGA and UT-PLGA/bFGF scaffolds was low. After culturing 10 days, theproliferation of cells on the PT-PLGA (20 W, 20 min) scaffoldwas higher than that on UT-PLGA/bFGF scaffold. Moreover,the cells on the PT-PLGA/bFGF scaffold showed the highestviability and the rapidest proliferation among various PLGAscaffolds.

2398 H. Shen et al. / Biomaterials 29 (2008) 2388e2399

Fig. 10 showed morphology of 3T3 fibroblasts on cross sec-tion of various PLGA scaffolds after cultured for 2 weeks. Inorder to clearly observe cells in the scaffolds, the morphol-ogies of scaffold before culture cells were shown as controls(Fig. 10a and b). It could be seen that there were only a fewcells, which gathered together and remained round shape, inthe pores of UT-PLGA and UT-PLGA/bFGF scaffolds(Fig. 10c and e). However, cells in the PT-PLGA (20 W,20 min) scaffold (Fig. 10d) could spread and grow alongpore wall. Especially in PT-PLGA/bFGF (Fig. 10f) scaffoldcells spread well and almost covered whole wall of pores.

Due to strong hydrophobicity and poor O-containinggroups of UT-PLGA scaffold, not only bFGF could not be im-mobilized on internal section of the scaffold, but also cellswere prevented from entering the internal section of the scaf-fold. So, only a little bFGF was coated on the surface of out-side pore of UT-PLGA scaffold and most cells gathered on thesurface of UT-PLGA and UT-PLGA/bFGF scaffolds. Since thebFGF on the UT-PLGA/bFGF scaffold could release rapidlyfrom the scaffold and there were not enough sites for cell rec-ognition and growth on the scaffold, the proliferation of cellson the UT-PLGA/bFGF scaffold was slow. However, since thebFGF on the UT-PLGA/bFGF scaffold could promote cellgrowth to some extent, proliferation and viability of cells onthe UT-PLGA/bFGF was higher than that on the UT-PLGAin the early culture time. Modifying depth of PLGA scaffoldby CO2 plasma treatment could reach to 4 mm under 20 Wof power and 20 min of treating time, which determined bya method of ink dye [45]. So, after CO2 plasma treatmentrich polar O-containing groups could be introduced onto sur-face of inside pores of the scaffold, which not only directly im-proved hydrophilicity of PLGA scaffold, but also providedmore sites to immobilize bFGF on surface of inside pores ofthe scaffold. The hydrophilicity could facilitate cells migrationinto the pores following culture medium when cells wereseeded into the scaffold. Because more cells migrated intothe pores of PT-PLGA (20 W, 20 min) scaffold and the effectof plasma treatment could be kept for relatively longer time,cell growth on the PT-PLGA (20 W, 20 min) scaffold waseven better than UT-PLGA/bFGF. In addition, it was more im-portant that higher amount of bFGF was immobilized evenlyon the PT-PLGA scaffold and sustained release of the immo-bilized bFGF could further promote cell growth. Thus, incontrast with PT-PLGA (20 W, 20 min) scaffold, cells on thePT-PLGA/bFGF scaffolds grew better. Thus, the PLGA scaf-fold modified by CO2 plasma treatment combining withbFGF anchorage could be utilized as a promising scaffoldfor tissue engineering.

4. Conclusions

In this study, it was evident that bFGF could be effectivelyimmobilized on the CO2 plasma-treated PLGA (PT-PLGA)scaffold. CO2 plasma treatment can increase the number ofO-containing functional groups and roughness of surface ofPLGA scaffold, which lead to effectively bind bFGF to surfaceof the PLGA scaffold. The binding bFGF efficiency of PLGA

was variation with CO2 plasma treating time and the best effi-ciency could be obtained by treating for 20 min under 20 W.The bFGF bound on the PT-PLGA film was stable and couldbe slowly released from the PLGA film for 7 days in vitro.The bFGF released from PT-PLGA scaffolds can maintain bio-activity. Furthermore, immobilization of bFGF on the PT-PLGA scaffold can improve adhesion and growth of cells onthe PLGA scaffold. Thus, the modified PLGA scaffold byCO2 plasma treatment combining with bFGF anchorage couldbe usable in delivering bFGF, and it could also be applied ex-tensively for surface modification of PLGA scaffold in tissueengineering. Moreover, this technique could be hoped to ex-tend to other growth factors and polylactone-type polymersby choosing suitable gas for the plasma treatment.

Acknowledgements

This research was supported by a grant from Major StateBasic Science Research and Development Program of China(973, No. 2005CB5227074) and High Technology Researchand Development Program of China.

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