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Biomaterials 28 (2007) 1838–1846

www.elsevier.com/locate/biomaterials

Time-dependent changes in adhesive force betweenchondrocytes and silk fibroin substrate

Koji Yamamotoa, Naohide Tomitaa,b,�, Yusuke Fukudac, Shingo Suzukid, Noboru Igarashie,Toru Suguroe, Yasushi Tamadaf

aDepartment of Mechanical Engineering, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Kyoto 606-8501, JapanbInternational Innovation Center, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Kyoto 606-8501, Japan

cDepartment of Mechanical Engineering, Graduate School of Engineering, Doshisha University, 1-3 Tataramiyakodani, Kyotanabe, Kyoto 612-0321, JapandDepartment of Engineering Science, Faculty of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto, Kyoto 606-8501, Japan

eDepartment of Orthopaedic Surgery, School of Medicine, Toho University, 6-11-1 Ohmorinishi, Ohta-ku, Tokyo 143-8541, JapanfNational Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan

Received 31 July 2006; accepted 29 November 2006

Available online 26 December 2006

Abstract

In tissue engineering for cartilage repair using scaffold, initial chondrocyte–material interactions are significantly important for the

following cell behaviors such as phenotypic expression and matrix synthesis. Silk fibroin scaffold is considered to be one of the useful

materials in/on which chondrocytes can proliferate without dedifferentiating into fibroblast-like cells and can organize a hyaline-like

tissue. For the purpose of seeking some useful aspects for designing scaffold, initial adhesive force of chondrocytes to the surface of

fibroin substrate was measured by using a lab-made apparatus applying the cantilever beam method. It was found that the adhesive force

per unit spreading area of chondrocytes on fibroin substrate had a clear peak between 6 and 12 h after seeding. From the results of

immunofluorescence staining for actin and vinculin during this period, it could be thought that an immature formation of actin fibers

which was uniquely observed at the periphery of cells attaching to fibroin substrate did not contribute to the increase of adhesive force.

Results in this study suggested that surface of the fibroin substrate was gradually covered with some substances which inhibit the

adhesion during this period. These cell–material interactions have a possibility to be useful information for designing the adhesive

performance of scaffold surface in cartilage regeneration.

r 2006 Published by Elsevier Ltd.

Keywords: Cell adhesion; Chondrocyte; Mechanical test; Silk

1. Introduction

In tissue engineering for articular cartilage repair, one ofthe problems is the difficulty in taking a lot of maturechondrocytes. Chondrocytes cultured on two-dimensionalculture plate proliferate in multiples, but the cells tend todedifferentiate into fibroblast-like cells, expressing type Icollagen not specific to intact cartilage [1]. Althoughchondrocytes embedded in three-dimensional gels, such as

e front matter r 2006 Published by Elsevier Ltd.

omaterials.2006.11.045

ng author. International Innovation Center, Kyoto University,

chi, Sakyo-ku, Kyoto, Kyoto 606-8501, Japan.

7539200.

ess: ntomita@iic.kyoto-u.ac.jp (N. Tomita).

collagen, agarose and fibrin can maintain cartilage-specificphenotype during in vitro culture [2–4], the proliferationability of them tends to be suppressed [5]. Therefore, one ofthe recent approaches in tissue engineering for cartilageregeneration has been how enough number of maturechondrocytes can be secured, applying various techniquesfor maintaining the phenotypic expression or for promotingthe redifferentiation of dedifferentiated cells [6–9]. Ourprevious studies showed that chondrocytes seeded into silkfibroin sponge could proliferate without dedifferentiationand a hyaline-like cartilage tissue was organized on thesurface of fibroin sponge as well as in the sponge [5,10].The mechanism why chondrocytes on fibroin substrate

can organize a hyaline-like tissue has not been clarified yet,

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but some initial adhesive conditions between chondro-cytes and the fibroin surface may modulate the chondro-genic performance. On the basis of effects of interactionbetween individual cells and substrate, the importancefor evaluating the phenomenon of cell adhesion, particu-larly cell adhesive force, has been recognized by variousinvestigators from the aspect of regenerative medicine[11–13]. Athanasiou et al. focused on the interactionbetween substrate proteins and mechanical adhesive-ness of an individual cell, developing the cytodetacher,which was able to measure the mechanical adhesivenessof a single cell using the actual shear force, andthey demonstrated that the adhesive force of chondrocyteson fibronectin substrate showed higher value than thaton glass substrate and on bovine serum albumin subs-trate [11], and it was found that the force per unitcontact area for glass substrate monotonically increasedbetween 0.5 and 6 h after seeding [12]. Lee et al.investigated the effect of enzymatic treatment forcartilage tissue using chondroitinase ABC on the adhesiveforce in the field of cell transplantation, and showed thatenzymatic treatment of a cartilage surface could enhancethe adhesion of chondrocytes [13]. These results couldexplain one of the characteristics on chondrocytes forvarious substrates and indicate some predictions for tissuegenesis. However, it would be still difficult to provide someinformation about a material surface suitable for cartilageregeneration.

Our motivation in this study is to seek useful aspects ofscaffold or substrate for cartilage regeneration, by inves-tigating initial adhesive performances of chondrocytes onfibroin substrate in consideration of above-describedfeatures. In the present study, glass substrate and fibroinsubstrate were prepared. Mechanical adhesiveness ofchondrocytes on each substrate cultured for up to 24 hwas measured by a newly developed apparatus, which isable to vertically pull a cell. Cell morphology was alsoobserved by immunofluorescence staining including actinconstruction and distribution of focal contacts.

Fig. 1. Photographs of chondrocytes on each substrate at 24 h afte

2. Materials and methods

2.1. Preparing protein-coated glass plates

The silk fibroin protein was coated on the tip of ultra-thin glass

plates (0100, Matsunami Glass Ind., Ltd., Japan) fabricated as the shape

of leaf spring (length: 45mm, width: 1.5mm, thickness: 30mm). The silk

fibroin aqueous solution was prepared from degummed silk fiber of a

Bombyx mori silkworm cocoon. The fibroin protein was refined by

dissolving its filaments in a 9.0M lithium bromide solution, and the diluted

organic solvent solution was gradually added to the fibroin aqueous

solution with stirring [14]. The fibroin aqueous solution contains the

fibroin proteins at the concentration of 0.5–1.0wt/vol% to be in saturated

solution. The ultra-thin glasses were soaked in this solution up to the point

of 15mm from the end of a glass and dried in a desiccator at 50 1C (FIB

group). Before the use of them, every leaf spring was extensively washed

with PBS. The glass without any coats was prepared as a control group

(CON group).

2.2. Preparing cells

Articular cartilage tissues were aseptically harvested from the

proximal humerus, distal femur and proximal tibia of 4-week-old Japanese

white rabbits (Oriental Bio Service Co., Ltd., Japan). The excised cartilage

tissues were diced into �1mm3 segments after adherent connective tissues

were removed from them, and chondrocytes were isolated via enzymatic

digestion. Small cartilage segments were digested with 0.25% trypsin-

EDTA (Nacalai Tesque, Inc., Japan) for 30min in a temperature-

controlled bath at 37 1C. After being rinsed twice with PBS and

centrifuged at 1500 rpm for 5min, they were enzymatically digested with

0.25% type II collagenase (CLS-2, Worthington Biochemical Co., USA)

solution prepared with Dulbecco’s modified Eagle medium (DMEM)

(Nacalai Tesque, Inc., Japan) not containing serum for 8 h at 37 1C. A

single-cell suspension was obtained, after being strained thorough

Cell strainer (BD FalconTM, USA) and washed twice with PBS. Cells

were cultured on T-flask (IWAKI Glass Co., Ltd., Japan) for 5 days, and

they were removed by trypsin-EDTA and seeded on the substrates put on

the bottom of a culture dish at a cell concentration of 5.0� 104 cells/ml.

The glass substrates on which cells were seeded were incubated with

DMEM containing 10% FBS and 1% antibiotics at 37 1C in 5% CO2.

After incubating them for 3, 6, 9, 12 and 24 h, the adhesive force of

individual chondrocytes to each substrate was evaluated. Fig. 1 displays

phase-contrast images of chondrocytes on each substrate at 24 h after

seeding ((a) CON group, (b) FIB group).

r seeding. (a) CON group, (b) FIB group. Scale bar ¼ 100mm.

ARTICLE IN PRESSK. Yamamoto et al. / Biomaterials 28 (2007) 1838–18461840

2.3. Apparatus for measuring the adhesive force

The main components of this apparatus were placed on an anti-

vibration table (SURUGA SEIKI Co., Ltd., Japan). Ultra-thin glass acted

as a cantilever, and a micropipette aspiration was used to hold a single cell

adhering to the substrate as shown in Fig. 2. In this study, adhesive force

was calculated using the amount of maximum deflection of the glass plate

observed through a video microscope (DG-2, Scalar Co., Japan)

connected to a time-lapse video recorder and the Young’s modulus of

the glass. The equation for the reaction force F is given by

F ¼ 3IEL�3o, (1)

where E is Young’s modulus (71.4GPa), I is moment of inertia of area, L

is the length from the fixed edge of a leaf spring to a cell and o is the

deflection at L. In this apparatus, it is possible to precisely determine the

value L, which can vary in accordance with the attachment position of a

cell, using the fine Z-axis positioner installed on the stage with the

micropipette. Prior to the use of a leaf spring, difference in the elastic

coefficient of the leaf spring between in presence and in absence of a

protein coating was evaluated by measuring the natural frequency. The

equation of the elastic coefficient k can be written as

k ¼ 4p2f 2m, (2)

where m is the mass and f is the natural frequency of the leaf spring. The

difference in the Young’s modulus between them was less than 1%. The

deflection in all experimental data was between 1.79 and 26.90mm.

Micropipettes were fabricated from glass capillaries (GC-1, NAR-

ISHIGE Co., Ltd., Japan) with outer and inner diameters of 1.0 and

0.6mm, respectively. The shape of the tip was processed into outer and

inner diameter of 30 and 4mm, respectively, by a micropipette puller

(NARISHIGE Co., Ltd., Japan) and a micro forge (NARISHIGE Co.,

Ltd., Japan). The opposite end of the micropipette was connected to a

manual microinjector (CellTrams Air, Eppendorf AG), which applied a

certain constant negative pressure to a cell. The pipette was installed on a

motorized X-axis stage (KS101-20HD, SURUGA SEIKI Co., Ltd.,

Japan) to control the moving velocity. The displacement rate of this stage

was set at 5 or 50mms�1. Cells were manipulated in a lab-made chamber

filled with Leibovitz’s L-15 medium (11415-064, GIBCO) containing 10%

FBS and 1% antibiotics, which allows cell culture independent of the

CO2 concentration. All of experiments were performed at between 34

and 37 1C.

Fig. 2. A schematic drawing of an apparatus for measuring the detachment for

by the micropipette which was able to hold a cell using negative pressure. The de

of the Y-axis in this image.

2.4. Procedure for the measurement of adhesive force

For each detachment measurement, one leaf spring on which

chondrocytes were cultured was fixed using the holder made of stainless-

steel in a clean bench and built in the apparatus, after incubated for 3, 6, 9,

12 and 24 h. In this experiment, the seeding time was staggered to avoid

more than 30min time lag at each time point. The number of specimens

which can be measured within 30min, was between 3 and 5. The

dependence of test duration and of experimental day in the adhesive force

was not found at each group. Both the leaf spring and the micropipette

were fully submerged into a chamber filled with pre-warmed L-15 medium,

and then the microscope was focused on a cell adhering to the substrate.

The leaf spring was positioned vertically so that the cells were oriented

perpendicular to the tip of the micropipette. The pipette was controlled

by the motorized positioning X-axis stage, until the tip touched the

upper surface of the cell. In this apparatus, it is possible to observe the

cells obliquely using the rotary stage installed under the microscope in

order to adjust the Y position. Negative pressure to hold a cell was then

applied by the microinjector, until a static hemispherical cap inside the

pipette was observed with the video monitor. The cell held by constant

negative pressure was pulled at the constant moving velocity of 5 or

50mms�1 (motorized stage velocity). Figs. 3(b1) and (b2) show video-

captured photographs of a single chondrocyte pulled by the pipette just

at the zero and at the maximum deflection point of the leaf spring,

respectively. Chondrocytes which were broken during the detaching

process were removed from the results. The number of data in each

group at each time was as follows: CON group; n ¼ 25, 30, 28, 23 and

25, FIB group; n ¼ 26, 31, 39, 40 and 27 at 3, 6, 9, 12 and 24 h,

respectively.

2.5. Measurement of cell spreading area

Cells were seeded on the two types of substrates as described

previously. After 3, 6, 9, 12 and 24 h, the cells were washed two

times with PBS and then fixed in 4% paraformaldehyde solution for

20min. After washing again in PBS, digital images of the cells were

acquired with a digital camera (C-5050 Z, Olympus Corp., Japan)

installed on a phase microscopy (IX71, Olympus Corp., Japan), and

then cell spreading area were measured by analyzing the binarized

images using Image J. First, a randomly selected area of the RGB image

ce of a single cell on a substrate. A single chondrocyte was vertically pulled

tachment process was recorded by the video microscope from the direction

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Fig. 3. A schematic drawing of holding a chondrocyte using micropipette aspiration (a) and photographs in the process of detaching a chondrocyte on

fibroin substrate at 9 h after seeding. (b1) o ¼ 0, (b2) o ¼ maximum deflection. Scale bar ¼ 50 mm.

K. Yamamoto et al. / Biomaterials 28 (2007) 1838–1846 1841

obtained by the digital camera was changed into a 8-bit-grayscale

image. Second, the background was subtracted from the 8-bit-gray-

scale image. Finally, the binary threshold was manually adjusted to

obtain the best contrast of the apparent cell shape, and other small

particles were manually removed. The same threshold value was used

for all groups, and these procedures were carried out by the same

operator.

Fig. 4. Cell spreading area on each substrate during adhesion process

from 3 to 24 h after seeding (CON; glass, FIB; fibroin). The data represent

the mean values and 7SD.

2.6. Immunofluorescence staining of actin and vinculin

The cells on the substrates were washed three times in PBS and

then fixed with 4% paraformaldehyde solution for 20min at room

temperature. After fixing, the cells were washed two times with a wash

buffer prepared with 0.05% Tween20 in PBS, and then permeabilized

in 0.1% Triton X-100 in PBS for 5min. After being washed twice with

the wash buffer, the cells were incubated in a fresh blocking solution

prepared with 1% BSA in PBS for 30min at room temperature, and then

anti-vinculin (MAB3574, CHEMICON International, Inc., USA) as a

primary antibody diluted with the blocking solution at 1:100 was added.

After 1 h, the cells were washed three times for 10min each time and then

incubated for 1 h, following the addition of FITC-conjugated secondary

antibody (AP124F, CHEMICON International, Inc., USA) at 1:100

dilution in PBS and of TRITC-conjugated phalloidin (R415, Molecular

Probes, USA). In a terminal process, the cells were incubated with DAPI

(S7113, CHEMICON International, Inc., USA) at 1:1000 dilutions to

distinguish a single chondrocyte, after being washed three times with the

wash buffer for 10min each time. The stained cells were observed with an

inverted fluorescence microscope, followed by a final wash with the wash

buffer.

2.7. Statistical methods

Effects of material interaction and culture time on adhesive force and

on spreading area were analyzed using two-way ANOVA followed by post

hoc comparisons. A value of po0.05 was considered significant.

3. Results

Fig. 4 represents the changes in cell spreading area as afunction of culture time. The cell spreading area on everysubstrate tends to increase depending on the increase ofculture time. Particularly, it was found that there wasstatistically significant difference in FIB group between 9and 12 h after seeding.The temporal fluctuations of the adhesive force are

displayed in Fig. 5. The results of monotonically increasingin cell adhesive force seemed to have the similar tendencyin the results exhibited by other researchers, even though

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the detaching direction was different [11,12,15]. In acomparison of the mean adhesive force, chondrocytes onfibroin substrate tended to produce greater resistive forcethan those on glass substrate between 6 and 12 h afterseeding.

The effect of cell’s viscosity involving the properties ofplasma membrane and other components in a cell wasexamined by pulling cells in the use of different pullingvelocity. Fig. 6 shows a comparison of the adhesive force inboth CON group and FIB group at 9 (Fig. 6(a)) and 24 h(Fig. 6(b)) after seeding, respectively. Regardless of thepulling velocity, both CON group and FIB group exhibited

Fig. 5. Adhesive force necessary to detach chondrocytes from different

substrates. In comparison of CON group and FIB group, there were

statistically differences at 6 and 9 h after seeding. The data represent the

mean values and7SD. Asterisks indicate statistical significance (*po0.05,

**po0.01, ***po0.001).

Fig. 6. Comparison of the adhesive force at the pulling velocity of 5 and 50mms

The data represent the mean values and 7SD. In both groups, there was no

each time.

a same tendency of the increase in adhesive forcedepending on culture time, and there was no statisticallysignificant difference in the comparison of the forcedepending on the pulling velocity at the same culture time.Transition of the adhesive force per unit spreading area

obtained by diving the mean adhesive force by the meanspreading area at each culture time showed quite differ-ences between groups as shown in Fig. 7. The result in FIBgroup clearly showed a peak between 6 and 12 h afterseeding and then decreased. Fig. 8 shows the transition ofthe adhesive force in a detaching process of both CONgroup and FIB group at 6 and 12 h, respectively. The datarepresent the detaching processes of the five results near to

�1 in both CON group (a) and FIB group (b) after 9 and 24 h, respectively.

statistically significant difference in the range between 5 and 50mms�1 at

Fig. 7. Adhesive force per unit spreading area as a function of culture

time. The data were obtained by dividing the mean adhesive force by the

mean spreading area at each culture time. Smoothing spline regressions

were applied for the curve fitting.

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Fig. 8. Transitions of the adhesive force in cell detaching processes of both CON group and FIB group at 6 h (a) and 12 h (b), respectively. The data

represent the detaching processes of the five results near to the mean adhesive force of all data at each time point. The X-axis represents a normalized

displacement of the motorized stage using the maximum moving distance necessary to cell detachment.

Fig. 9. Immunofluorescence staining of F-actin (a, b, e, f) and vinculin (c, d, g, h) in chondrocyte on glass substrate (a–d) and on fibroin substrate (e–h) at

6 and 12 h after seeding, respectively. Scale bar ¼ 20 mm.

K. Yamamoto et al. / Biomaterials 28 (2007) 1838–1846 1843

the mean adhesive force of all data at each time point. TheX-axis represents a relative moving distance of themotorized stage with micropipette, in which cell deforma-tion was included and the true distance was normalized bythe maximum moving distance necessary to cell detach-ment. It was found that FIB group varied more linearlywith relative displacement than CON group even at 6 h,which means that the result of CON group would be moreaffected by cell deformation than that of FIB group.

Immunofluorescence staining revealed distinct differentpatterns between CON group and FIB group in compar-ison of 6 and 12 h after seeding. Photographs displayed inFig. 9 show the representative pattern of actin construc-tions and vinculin distributions under each condition.Chondrocytes on FIB group showed better expression ofvinculin at 6 h after seeding. However, actin and vinculin

appeared to be eccentrically located at the central portionin a cell at 12 h after seeding. On the other hand, in the caseof chondrocytes on CON group, vinculin was expressed atthe peripheral portion in a cell at 6 h after seeding, butat 12 h, dash-like focal contacts were uniformly observed ina cell.

4. Discussion

As shown in previous reports [5,10], one of the featuresin using fibroin sponge as a scaffold is that a hyaline-likecartilage tissue can regenerate on the surface of the sponge,and chondrocytes can also easily proliferate compared withcollagen gel. In this study, initial interactions amongchondrocyte and fibroin surface were investigated to seeksome useful information on designing a scaffold surface for

ARTICLE IN PRESSK. Yamamoto et al. / Biomaterials 28 (2007) 1838–18461844

an efficient tissue regeneration. The results in the adhesiveforce per unit spreading area revealed that chondrocytes onfibroin substrate clearly had a peak and then decreasedbetween 9 and 12 h. On the other hand, that ofchondrocytes on glass substrate gradually increased duringthis period. In a comparison of the results demonstrated byHuang et al. [12], the spreading area of chondrocytes onglass substrate at 6 h in our results was quite larger thanthose in their results. In our experiments, glass substrateswere used after being washed two times with PBS. Thisprocedure might alter the hydrophilicity and/or hydro-phobicity of the substrate surface. However, cell–materialadhesion can be also influenced by the culture conditions,including the age of the animal and removal portions oftissue. From the results of immunofluorescence staining,the localization of actin polymerization was not found atthe leading edge at 12 h in FIB group, but fairly dispersionof dot-like vinculin was observed along the periphery ofcell body. This appearance would be one of the reasonswhy the increase of the spreading area could not contributeto the increase of the adhesive force in FIB groupcompared to CON group. In cell adhesion phenomena,intermediate adherence state characterized by a spread cellthat lacks actin-containing stress fibers terminating atadhesion plaques is considered to have a potential tomediate morphogenesis [16]. Additionally, the process ofcytoskeleton organization based on the actin microfila-ments has been demonstrated to correlate with not only theproduction of mechanical force but also the state ofphenotype [17,18]. Therefore, it could be thought thatchondrocytes on fibroin substrate expressed dynamicmembrane changes during this period which relate to thesignal transduction of phenotypic expression and/orsynthesis of extracellular matrix proteins, conducting theformation and the disassembly of both cytoskeletalstructure and focal contacts. These specific cascadesexhibited in FIB group are likely to be affected by theinteraction between adhesive proteins or secretions ofchondrocytes and the properties of fibroin surface.

When comparing the transition of adhesive force in thedetaching process of FIB group and of CON group at both6 and 12 h, it was found that the resistive force ofchondrocytes on fibroin substrate tended to linearlyincrease at both culture time. Although these resultscontain the deformation of cell membranes, the distribu-tion pattern of focal contacts is considered to be animportant factor to affect cell’s mechanical behaviors. Theexpression of vinculin in CON group at 6 h tends tocolocalize with well synthesis of actin fibers at theperiphery of cell body, and the cell of which the centralportion has immature actin stress fibers and low-densitydistribution of focal contacts could be easily deformed by avertical pulling force. When considering the result that thefocal contacts expressed in chondrocytes on fibroinsubstrate at 6 h appeared to be uniformly distributed, theresult in FIB group could explain a facilitation of a stableadherence, not meaning a strong adherence, from the early

phase in cell attachment compared to those on glasssubstrate. However, between 6 and 12 h after seeding, theattachment in the peripheral portion of cells to fibroinsubstrate seemed to be inhibited by some mechanismsrelating to the chondrocyte–fibroin interaction. One of thepossibilities is a change in the interaction among fibroinsubstrate and the adhesion proteins, mainly fibronectin andvitronectin contained in the culture solution. Particularly,binding of adhesion receptors to adsorbed fibronectinprovides mechanical coupling to the substrate and theconformation of adsorbed fibronectin which results inmechanical coupling to the ligand can be altered bysubstrate properties [19]. Furthermore, these conforma-tional changes in fibronectin can affect its biologicalactivity [20–22] and a modulation of cell proliferationand differentiation [23]. Tamada et al. reported the kineticchange in the properties of silk fibroin surface from theaspect of crystallization [14]. These might result in thespecific changes in the binding of fibronectin to domains onfibroin substrate.Another possibility is the effect of substances which

chondrocytes synthesized, since the specific extracellularmatrices (ECMs) can regulate the behavior of integrinexpression which influences various physiological andbiological properties of cells. In particular, functions ofa5b1 integrin as a fibronectin receptor are involved in celladhesion, proliferation and differentiation in chondrocytes[24–26], and the expression of the integrin can be affectedby various factors, such as the surface roughness [27],mechanical stimulations [28] and ECMs [24]. Imoto et al.showed that when USAC cells adhering to a substratecoated with fibronectin were cultured with a-MEMcontaining exogenous proteoglycan, the expression of b1integrin had diminished between 12 and 24 h after addingthe proteoglycan [29]. The result means that the proteogly-can could regulate the expression of integrin and inhibit thecell adhesion. Additionally, aggrecan synthesized bychondrocytes has a high negative charge density due tochondroitin and keratan sulfate chains. Hence, theglycosaminoglycan chains in proteoglycan might causeelectric repulsion through ionic interactions with thefibroin surface which has a negative charge. In our results,although the adhesive force of chondrocytes on fibroinsubstrate did not exhibit the decrease with the increase ofculture time, the adhesive force per unit area oncedecreased at the early adhesion phase. These results mayindicate that proteoglycan synthesized by chondrocytes onfibroin substrate acted as a protein which inhibit celladhesion and can affect the distribution patterns of focalcontacts.It must be important to analyze the expression of

integrins and to investigate the synergistic interactionbetween silk fibroin substrate and adhesive proteins,particularly adsorbed fibronectin, in our future studies.It is also necessary to consider the effect of the detach-ment direction. When using shear force, it would be easyto observe the cell detachment processes dynamically

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measuring the detachment force. One of the reasons on theadoption of this methodology applying pulling force ismainly due to a difficulty in identifying the true surface offibroin substrate. In addition, shear force would beinfluenced by a relation between the direction of movementon cantilever and the arrangement of the actin fibers or thedistribution of the focal contacts. Although our apparatushave some problems such as the procedure of holding cells,this methodology would be useful for analyzing ormodeling changes in the stress distribution in adhesivecells during the detachment process.

5. Conclusions

In this study, initial attachment properties of chondro-cytes on silk fibroin substrate were investigated using a lab-made apparatus for measuring the adhesive force. Bothadhesive force and spreading area of chondrocytes on allsubstrates tended to monotonically increase with theincrease of culture time. However, the adhesive force perunit spreading area of chondrocytes on fibroin substrateexhibited a peak between 6 and 12 h after seeding. Inaddition, the formation of cytoskeleton and the distribu-tion of focal contacts were also affected by the interactionwith the fibroin surface. Results of this study will providethe useful information to analyze the process of tissueorganization and to design the surface properties of ascaffold for cartilage regeneration.

Acknowledgments

This paper was supported in part by Knowledge ClusterInitiative (MEXT, Kyoto Nanotech Cluster), Japan HealthScience Foundation (MHLW, K7200001-01), Nanotech-nology Project (MAFF, Development of nanotechnologyand materials technology for innovative utilization ofbiological functions, III-2-2) and Center of Excellence forResearch and Education on Complex Functional Mechan-ical Systems (COE program of the Ministry of Education,Culture, Sports, Science and Technology, Japan).

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