incorporation of basic fibroblast growth factor by a layer-by-layer assembly technique to produce...

Incorporation of Basic Fibroblast Growth Factorby a Layer-by-Layer Assembly Technique to ProduceBioactive Substrates

Lie Ma, Jie Zhou, Changyou Gao, Jiacong Shen

Key Laboratory of Macromolecular Synthesis and Functionalization, Ministry of Education, Department of PolymerScience and Engineering, Zhejiang University, Hangzhou 310027, China

Received 30 April 2006; revised 5 January 2007; accepted 10 January 2007Published online 23 March 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30794

Abstract: Basic fibroblast growth factor (bFGF) was immobilized onto quartz slides and

collagen films by assembly with chondroitin sulfate (CS) in a layer-by-layer (LBL) manner.

First, the LBL-deposition process on the amino-silanized quartz slides was monitored by UV–

vis spectroscopy and water contact angle measurement. By substituting the normal bFGF with

rhodamine-labeled one (Rd-bFGF), a linear increase of the absorbance versus bilayer number

was recorded. The water contact angle oscillated between the odd CS and the even bFGF

layers, demonstrating the alternating change of the surface chemistry. Scanning force

microscopy (SFM) revealed that the surface topography was altered slightly after multilayer

assembly. In vitro incubation of the CS/bFGF multilayers in PBS showed that *30% of the

incorporated bFGF was released within 8 days. In vitro cell culture found that the fibroblasts

showed star-like morphology with plenty of pseudopods on the bFGF-incorporated collagen

film after cultured for 1 day, and the collagen films assembled with bFGF possess improved

bioactivity than that of the virgin one and the bFGF control. Since the immobilized growth

factors can maximally retain their bioactivity, the LBL assembly would be a potential

approach to construct a bioactive substrate for biomedical applications. ' 2007 Wiley Periodicals,

Inc. J Biomed Mater Res Part B: Appl Biomater 83B: 285–292, 2007

Keywords: layer-by-layer; cell-materials interaction; interface; growth factor; collagen

INTRODUCTION

Tissue engineering is emerging as a useful strategy for

replacement and regeneration of damaged organs or tis-

sues.1,2 Nowadays, it is regarded as one of the most prom-

ising ways in tissue engineering to induce the natural

regeneration mechanism of the body. For this purpose,

incorporation of various signal generating molecules is gen-

erally adopted, since the signals can regulate cells’ prolifer-

ation and differentiation in the body.3–5 These signals can

be generated from growth factors, cell-extracellular matrix

(ECM), and cell–cell interactions, as well as from physi-

cal–chemical and mechanical stimuli.6,7

It is known that various growth factors are always

involved in tissue regeneration. They can regulate prolifera-

tion and differentiation of different types of cells in the tis-

sue healing process. Therefore, many efforts have been

devoted to constructing bioactive substrates by incorporation

of growth factors related to tissue regeneration. Among

which basic fibroblast growth factor (bFGF) has been widely

applied in the regeneration of bone,8 cartilage,9 skin,10 and

nerve.11,12 In human body, bFGF always binds with ECM

components such as polysaccharides by intermolecular inter-

actions. When simply added into solution, however, the

bFGF will lose its bioactivity quickly. Therefore, many car-

rier systems such as gels and microparticles have been

developed to deliver the growth factors.13,14 Control over

the concentration, local duration and spatial distribution of

the growth factors are the key factors for their utility and ef-

ficacy. Recent advances have been made in developing poly-

meric delivery systems to achieve this control.15,16

In the past decades, layer-by-layer (LBL) assembly ini-

tially introduced by Decher has been developed rapidly and

broadly.17 The principle of the LBL assembly technique is

based on the alternate adsorption of polyelectrolytes with

opposite charge. Because of the rich choice of the

Correspondence to: C. Gao (e-mail: [email protected])Contract grant sponsor: Major State Basic Research Program of China; contract

grant number: 2005CB623902Contract grant sponsor: Ph.D. Programs Foundation of Ministry of Education of

China; contract grant number: 20050335035Contract grant sponsor: Natural Science Foundation of China; contract grant

number: 20434030Contract grant sponsor: National Science Fund for Distinguished Young Scholars

of China; contract grant number: 50425311

' 2007 Wiley Periodicals, Inc.

285

assembled polyelectrolytes and less restriction of assembled

substrate, this technique has been extended to assemble dyes,

conducting polymers, and biological macromolecules.18–20

More recently, attempts have been made to seek practical

applications too, particularly in biomedical fields. For exam-

ple, LBL assembly of ECM components on synthetic poly-

mers was used to create a cytocompatible interface.21

Moreover, polysaccharides, nucleic acids, and functional

peptides are all employed in modification of biomaterials by

the LBL technique.22,23 Yet, incorporation of cell growth

factors by the LBL technique is rarely reported so far.

The purpose of this work is to explore the feasibility of

immobilizing cell growth factors via the LBL assembly

technique. Exemplified here with bFGF (positively charged

at neutral pH), the incorporation is accomplished by inter-

action with a negatively charged polysaccharide, chondroi-

tin-4-sulfate (CS). To monitor the LBL-deposition process,

amino-silanized quartz slides are first adopted for their high

transparency in UV–vis region. Assembly of bFGF and CS

is then extended to collagen films to seek a practical appli-

cation. Accordingly, the bioactivity of the modified colla-

gen substrates is evaluated by in vitro cell culture.

MATERIALS AND METHODS

Materials

Collagen type I was isolated from fresh bovine tendons by

a trypsin digestion and acetic acid extraction method.

Recombinant bFGF (freeze-dried powder, 500 U/mg) was

obtained from Changchun ChangSheng Gene Pharmaceuti-

cal (China). CS and trypsin (250 U/mg) were purchased

from Sigma (USA). All other reagents and solvents were of

analytical grade and used as received.

Rhodamine-labeled bFGF (Rd-bFGF) was prepared by

incubating the bFGF solution in 0.2 mg/mL rhodamine B

isothiocyanate at 48C for 48 h. The unreacted dyes were

dialyzed off in distilled water for 4 weeks. The dialyzing

solution was changed every 3 days.

Preparation of Substrates

For the sake of monitoring the assembly process, the CS/

bFGF multilayers were first constructed on quartz slides.

Before assembly, the quartz slides were cleaned in a

freshly prepared \piranha" solution (7:3 concentrated 98%

H2SO4:30% H2O2) for 5 min, and then were rinsed exten-

sively with water. The cleaned quartz slides were treated in

2% (v/v) c-aminopropyltriethoxysilane (APTES)/95% etha-

nol solution for 20 min, and then were dehydrated at

1108C for 1 h to obtain the amino-silanized quartz slides.

The collagen films were prepared by casting 1% (w/v)

collagen/0.5% acetic acid solution on a stainless steel plate

and then dried at 258C for 48 h. The films were further

treated at 1058C under reduced pressure (<0.2 mbar) for

another 24 h.

LBL-Deposition Process

First, all the substrates were incubated into 3% (w/v) acetic

acid solution for 15 min to obtain the positively charged

surfaces. Then, the substrates were immersed into CS solu-

tion (1 mg/mL with 0.1M NaCl, pH 5.6) for 15 min at

room temperature, followed by rinsing with 0.1M NaCl so-

lution for three times. Subsequently, the substrates were

dipped into bFGF/PBS solution (10 mg/mL) for 15 min, fol-

lowed by rinsing with PBS (pH 7.4) for three times. Fur-

ther deposition of the CS/bFGF on the substrates was

accomplished by repeating the same cycle as shown in

Figure 1. The cycle was repeated for n times to obtain

substrates with (CS/bFGF)n multilayers.

Characterization

The deposition process of the CS/bFGF multilayers on the

quartz slide was monitored with a UV–vis spectrophotome-

ter (CARY 100 BIO, America) by substituting the normal

bFGF with Rd-bFGF. Oscillation of water contact angle

between layers was measured on a DSA10-MK2 contact

angle measuring system from Kruss in a sessile drop man-

ner. The morphology of the multilayers on the quartz slides

was characterized with a scanning force microscope (SFM)

(SPI3800N, Seiko) in a dynamic force mode. The cross-

section image of the collagen film assembled with (CS/

Rd-bFGF)10 multilayers was recorded with a confocal laser

scanning microscope (CLSM) (Bio-Rad Radiance 2100).

Fibroblast Isolation and Culture

Fibroblasts used in this study were isolated from human

foreskin by collagenase digestion. Briefly, the epidermis

and subcutaneous tissue of human foreskin were removed

by a scalpel. The residual dermis was diced into 0.5–

1 mm3 sized tissues, and washed with PBS supplemented

Figure 1. Schematic illustration to show the layer-by-layer assembly

of chondroitin sulfate (CS) and basic fibroblast growth factor (bFGF)

on an amino-silanized solid substrate such as quartz slide and col-lagen film. [Color figure can be viewed in the online issue, which is

available at www.interscience.wiley.com.]

286 MA ET AL.

Journal of Biomedical Materials Research Part B: Applied BiomaterialsDOI 10.1002/jbmb

with penicillin (100 U/mL) and streptomycin (100 U/mL)

for three times. Then, these dermis pieces were placed in a

spinner flask (Falcon) containing 10 mL 1 mg/mL collage-

nase (type I, Sigma) in Dulbecco’s modified Eagle medium

(DMEM, Gibco) supplemented with penicillin (100 U/mL)

and streptomycin (100 U/mL). After the dermis pieces were

digested in an incubator (378C, 5% CO2) for 5 h, suitable

amount of DMEM supplemented with penicillin (100 U/mL),

streptomycin (100 U/mL), and 10% fetal bovine serum

(FBS) (complete medium) was added to terminate the diges-

tion. The digesting solution was filtered through a copper

mesh (cell strainer, 200 meshes), and then was centrifuged at

1000 rpm for 10 min. After discarded the supernatant, com-

plete medium was added to suspend the cells. The fibroblasts

were cultured in culture plates at 378C, 5% CO2, and 95%

humidity. The culture medium was changed every 3 days.

Cell Morphology

Cells were passaged at confluence and fourth to eighth pas-

sage fibroblasts were used to evaluate the bioactivity of the

collagen films. After cell-seeding for 1 and 7 days, the vir-

gin collagen films and the (CS/bFGF)5-modified collagen

films were washed with PBS for two times, and then were

treated in 5 mg/mL fluorescein diacetate (FDA) solution for

15 min. Only viable cells could metabolize FDA (nonfluor-

escent) to fluorescence probe. After removal of the

unreacted FDA by two washings in PBS, 1 mL complete

medium was added. Fluorescent images of the viable fibro-

blasts were recorded by CLSM.

Cell Attachment and Proliferation Behaviors

All the samples were sterilized with ethylene oxide before

cell seeding. The films were placed on the bottom of a well

of 96-well tissue culture polystyrene plate (TCPS, Falcon).

Into each well, 5000 human dermal fibroblasts were seeded.

Meanwhile, a blank TCPS well was seeded with the same

number of cells and supplemented with 0.2 mg bFGF (bFGF

control). The number of fibroblasts on different samples was

quantified by a cell-counting method. Briefly, the cells at

each interval were digested by 0.25% (w/v) trypsin for 10–15

min at 378C. The cell number was then counted under a hae-

macytometer. In these experiments, no serum was used. The

culture medium (without serum) was changed every 3 days.

The cell attachment ratio was defined as the ratio of the

remaining cell number on the substrate after 12 h culture to

the initial number. The cell-proliferation behavior was eval-

uated by examining the time-dependent change of fibro-

blast number. Each value was averaged from three parallel

measurements.

Statistical Analysis

Data are expressed as the mean 6 SD. Statistical analysis

was performed using two-population Student’s t test. The

significant level was set as p < 0.05.

RESULTS

The LBL-Deposition Process

The change of absorbance of the rhodamine labeled bFGF

as a function of layer number is presented in Figure 2. It

shows that the absorbance of the multilayers increased

almost linearly with the layer number, demonstrating that

bFGF was surely assembled on the slide in a LBL manner.

Figure 3 shows the alternation of water contact angle as a

function of layer number. The samples with odd and even

number represent CS and bFGF as the outmost layer,

respectively. Basically, the water contact angles oscillated

between 558–608 (CS as the outmost layer) and 658–708(bFGF as the outmost layer). This result implies that the

CS layer is more hydrophilic than the bFGF layer, and

indicates that they are deposited in a LBL manner.

Figure 2. Absorbance (*560 nm) of Rd-bFGF in the CS/Rd-bFGF

multilayers as a function of the layer number.

Figure 3. Water contact angle on the assembled substrate as a

function of layer number. Odd and even numbers represent CS andbFGF as the outmost layer, respectively.

287INCORPORATION OF bFGF BY A LAYER-BY-LAYER ASSEMBLY TECHNIQUE


The change of the surface topography before and after

LBL deposition was detected by SFM (Figure 4). The

untreated quartz slide shows a relative smooth topography

with an average surface roughness (RMS) of 0.68 nm

[Figure 4(a)]. After treated with APTES, tiny dots emerged

on the quartz surface, leading to a rougher surface with an

average RMS of 0.94 nm [Figure 4(b)]. These dots could

be the product of inhomogeneous self-crosslinking of

APTES. After assembled with one layer of CS, the amino-silan-

ized surface became smoother (RMS 0.81 nm) [Figure 4(c)],

implying the occurrence of surface coating. After deposi-

tion of five bilayers, a more irregular and rougher surface

(RMS 1.1 nm) was obtained [Figure 4(d)].

Stability of the Assembled Multilayers

To detect the stability of the incorporated bFGF, a quartz slide

deposited with 10 bilayers of CS and Rd-bFGF was immersed

into PBS (pH 7.4). The absorbance from the quartz slide was

then recorded as a function of time by UV–vis spectroscopy

(Figure 5). It shows that in the early 4 h, no significant

decrease of absorbance had been measured. After immersed

for 12 h, the absorbance reduced *8%, demonstrating that

the CS/bFGF multilayers have suitable stability in the initial

stage. A more quick decay of the absorbance occurred

between 12 and 48 h. The cumulative release amount of

bFGF reached to about 20% at 48 h. Then, the bFGF in the

multilayers could be released up to 8 days at a relatively slow

rate. Meanwhile, the topography change after immersion in

PBS was also examined as shown in Figure 6. The multilayer

surface lost their irregular topography gradually and became

smoother with the prolongation of immersion time.

Evaluation of the Bioactivity

Figure 7 shows the cross-section of the collagen film

assembled with 10 bilayers of CS/Rd-bFGF, which was

observed directly by CLSM. It shows that there exists a red

Figure 4. SFM images to show the quartz slide surface (a), amino-silanized quartz slide surface (b),

surfaces assembled with one layer of CS (c), and five bilayers of CS/bFGF (d). [Color figure can beviewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 5. Absorbance (*560 nm) of (CS/Rd-bFGF)10 multilayers as

a function of time. The multilayers deposited on a 1 3 2 cm2 quartzslide were immersed in PBS at room temperature.

288 MA ET AL.


ultrathin layer on each side of the film, which is exclu-

sively attributed to the emission of the Rd-bFGF.

Cell Morphology. The morphology of the fibroblasts

seeded on different substrates for different time was com-

pared (Figure 8). The images of 1 day show that the fibro-

blasts could adhere and spread on all the substrates. However,

the fibroblasts on the virgin collagen film exhibit polygonal

morphology with few pseudopods. By contrast, the cells on

the bFGF incorporated substrate show the star-like morphol-

ogy with plenty of pseudopods, indicating that the cells might

be in the state preparing for differentiation [Figure 8(a,c)]. Af-

ter 7 days of culture, more cells with typical fibroblast mor-

phology were observed on the bFGF-assembled substrate

than that on the unmodified one [Figure 8(c,d)].

Cell Attachment and Proliferation Behaviors. The

attachment ratios of fibroblasts on different kinds of sam-

ples are compared in Figure 9. No significant difference

(p > 0.05) was found between these samples. Figure 10

compares the change of the cell number on these samples

with the culture time. The fibroblasts proliferated more rap-

idly at the first day on the (CS/bFGF)5 multilayer modified

film and the bFGF control than on the virgin collagen film

(p < 0.05). Although no significant difference was found

between the number of cells cultured on the (CS/bFGF)5multilayer modified film and the bFGF control. At day 2,

more fibroblasts was detected on the (CS/bFGF)5 multilayer

modified film than that on the bFGF control and the virgin

collagen film. Although the cells continued dividing, no

significant difference was found for all the samples

between day 2 and day 4. After day 4, the cell number still

kept at a relatively high level without obvious change.

DISCUSSION

In this work, the bioactive substrates were constructed by

incorporation of bFGF in a LBL manner. First, the deposi-

Figure 6. SFM images to show the surface topography of the (CS/bFGF)10 multilayers before (a)and after the multilayers were immersed in PBS (pH 7.4) for (b) 2, (c) 5, and (d) 7 days. [Color figure

can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 7. CLSM image to show cross-section of the collagen film

assembled with 10 bilayers of CS/Rd-bFGF. [Color figure can be viewedin the online issue, which is available at www.interscience.wiley.com.]



tion process was monitored by the UV–vis spectroscopy to

testify the realization of the LBL assembly. In consideration

of the poor transparency of the collagen films in UV–vis

region, quartz slides were used to monitor the LBL growth

process of CS and bFGF. In addition, to record the absorb-

ance change of bFGF with the deposition process, the nor-

mal bFGF was substituted by Rd-bFGF, which shows the

maximum absorbance at about 560 nm. The linear increase

of the Rd-bFGF absorbance with the layer number proved

that bFGF has been surely incorporated on the slide in a

LBL manner. It is worth mentioning that the bFGF amount

in the first bilayers was somewhat larger than that of the

latter bilayers. This could be caused by additional physical

adsorption besides the charge interaction.

In general, surface wettability of a material is mainly

decided by its superficial layer. Therefore, the oscillation of

the surface wettability caused by the different building

blocks (either CS or bFGF as the outmost layer) can be con-

veniently tracked by water contact angle, and accordingly

can be used to prove the LBL-deposition process. Since

bFGF is an amphiphilic protein, all the samples were dried at

378C for 3 h before the measurements. This would benefit

the hydrophobic domains of bFGF to expose to the layer sur-

face, so that larger difference of wettability can be achieved.

The surface morphology of the material is one of the

key aspects determining the cell behaviors. Therefore, the

surface morphology after deposition should be examined.

The SFM images demonstrated that, in general, the modi-

fied surface is very smooth and the topography change of

the quartz surface is very minimal. Thus, one can exclude

the influence of surface morphology on the cell behaviors.

The stability of the assembled multilayers against incu-

bation is crucial for the growth factor to perform biological

function. Multilayers with low stability will dissociate

quickly and the bioactivity of the growth factor will be rap-

idly lost. On the other hand, the growth factor in a bioac-

tive substrate should also be released in a suitable manner

to exert necessary biological response. The PBS-immersion

test showed that the release of the incorporated bFGF taken

place mostly in the period from 12 to 48 h, in which the

fibroblasts are commonly in the proliferation stage. Then,

the remaining bFGF can be released steadily for up to 8

days. This sustaining release could accelerate the cells pro-

liferation in a relatively long time.

The bioactivity of the bFGF in the as-prepared multi-

layers was assessed by in vitro fibroblast culture. For this

Figure 8. CLSM images to show the viable fibroblasts on the virgin

collagen films (a,c) and the (CS/bFGF)5-modified collagen films (b,d)

after cultured for 1 day (a,b) and 7 days (c,d). Viable cells were

stained green (bright color) with fluorescein diacetate (FDA). [Colorfigure can be viewed in the online issue, which is available at

www.interscience.wiley.com.]

Figure 9. Cell attachment ratio on virgin collagen film, bFGF control

(0.2 mg/well), and (CS/bFGF)5 multilayer modified collagen film. 96-well TCPS was used and the cells were cultured for 12 h.

Figure 10. In vitro cell proliferation on virgin collagen film, bFGFcontrol (0.2 mg/well), and (CS/bFGF)5 multilayer modified collagen

film as a function of culture time. *Indicates p < 0.05.

290 MA ET AL.


purpose, instead of quartz slide, collagen film was

employed as substrate to assemble bFGF, since this mate-

rial has a wide application in biomedical field. However, it

was hard to monitor the LBL process directly on the colla-

gen film by methods such as UV–vis spectroscopy because

of the translucence of the collagen film. Nevertheless, we

would expect similar multilayer structure on the collagen

film. Therefore, CLSM was performed to observe directly

the cross-section of the collagen film deposited with CS/

Rd-bFGF multilayers. It has to note that it is not reasonable

to judge the multilayer thickness from the CLSM image,

because the best resolution of CLSM is practically larger

than 300 nm at an excitation wavelength of 543 nm.

Although the layer structure and thickness cannot be

derived from this observation, the incorporation of bFGF is

undoubtedly confirmed.

Normally, the interaction processes between cells and

substrate can be divided into several stages such as adher-

ing stage, spreading stage, proliferation stage, and so forth.

Hence, the cell attachment and proliferation behaviors are

the key factors to determine the function of the incorpo-

rated bFGF. There has no significant difference of the cell

attachment ratio between the virgin collagen film and the

bFGF-modified one. The result is reasonable, because

bFGF has, in most cases, no positive effect on the adhering

performance of fibroblasts.24

Finally, the cell-proliferation behaviors were recorded

by measuring the cell number on the different substrates

with the culture time. On the basis of the similar cell

attachment ratio, the cell number at each interval can repre-

sent the cell-proliferation behavior. From the results of the

cell-proliferation test, one can conclude that (1) in the cul-

ture medium absence of serum, the supplement of bFGF,

regardless of its form, has positive effect on fibroblast pro-

liferation than the virgin collagen film, in particular, in the

initial stage; (2) the LBL-immobilized bFGF has a stronger

ability to accelerate the proliferation of fibroblasts than the

unimmobilized one. Taking into account the typical thick-

ness of a single layer (around 2 nm), the total amount of

bFGF in the (CS/bFGF)5 multilayers (double sides on the

collagen film) is estimated as 0.5 mg. According to Figure 5,

the released amount of bFGF is around 20%. Hence, the

maximal soluble amount of bFGF is *0.1 mg, only 50% of

the bFGF control. One can then further conclude that the

LBL immobilization can largely preserve the bioactivity of

the cell-growth factor.

CONCLUSIONS

Layer-by-layer assembly technique is successfully employed

to construct CS/bFGF multilayers on quartz slides and colla-

gen films. The linear increase of the absorbance and the oscil-

lation of contact angle between even and odd layers have

confirmed that the multilayers are constructed in a LBL man-

ner. The deposited CS/bFGF multilayers can be partly disas-

sembled to release the deposited bFGF at a proper rate. The

cell-proliferation test and the cell morphology observation

demonstrate that the collagen films assembled with bFGF

possess an improved bioactivity than that of the virgin one

and the bFGF control, demonstrating that the LBL immobili-

zation can largely preserve the bioactivity of the cell-growth

factor. Thus, it is regarded as a promising approach to incor-

porate cell-growth factors, which can be extended to modify-

ing 3D scaffolds too and is under investigation now.

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