Biomaterials 24 (2003) 37253730

Chondrocyte behaviors on poly-l-lactic acid (PLLA) membranescontaining hydroxyl, amide or carboxyl groups$

Zuwei Ma, Changyou Gao*, Yihong Gong, Jiacong Shen

Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

Received 26 October 2002; accepted 29 March 2003

Abstract

Hydrophilic groups, i.e. hydroxyl (OH), carboxyl (COOH) or amide (CONH2) were introduced onto the poly-l-lactic acid(PLLA) membrane surfaces via the photo-induced grafting copolymerization of the corresponding monomers, i.e. hydroxyethyl

methacrylate, methacrylic acid or acrylamide, respectively. Chondrocyte culture was used to study the correlation between the cell

behaviors and the hydrophilic functional groups. The results showed that the cytocompatibility of the PLLA membranes with

hydroxyl or amide groups on the surface was greatly improved compared to that of the original PLLA membrane. However, the

PLLA membrane with carboxyl groups on the surface had even worse cytocompatibility though possessed a similar hydrophilicity.

r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Poly-l-lactic acid (PLLA); Hydrophilicity; Cytocompatibility; Surface modication; Chondrocyte

1. Introduction

It has been known that both the chemistry and thetopography of biomaterial surface may directly inu-ence the cell behaviors through adsorbing the extracellular matrix (ECM) molecules and altering theirconformation that, in turn, regulate cellsubstrateinteractions [13]. Surface characteristics such as hydro-philicity [49], surface charge density [10], surfacemicromorphology [1113], free energy [14] and specicchemical groups [15] affect the cell adhesion, spreadingand signaling, and hence regulate a wide variety ofbiological functions, including cell growth, cell migra-tion, cell differentiation, synthesis of extracellularmatrix and tissue morphogenesis.Hydrophilicity is one of the most important factors

that affect the cytocompatibility of biomaterials. Theadhesion and growth of cells on a surface are consideredto be strongly inuenced by the balance of hydrophili-city/hydrophobicity, frequently described as wettability[1619]. Although it has been reported that there was no

obvious correlation between the wettability and cellbehaviors [2022], many works have demonstrated thatcells prefer to attach on hydrophilic surface than onhydrophobic surface [4,5,7,9,1619]. Further studiesfound that cells adhered, spread and grown more easilyon substrates with moderate hydrophilicity than onhydrophobic or very hydrophilic substrates [4,6,8]. Cellmigration rate was also affected by the surface hydro-philicity, which linearly increased in reverse to the sessilecontact angle (SCA) but reduced if the hydrophilicsurface had a mobile chemistry [23]. Because surfacehydrophilicity plays such important roles in cytocom-patibility of biomaterials, many methods such as plasmatreatment in ammonia or sulfur dioxide [2425],irradiation, photo- or plasma-induced grafting ofhydrophilic polymers [2629] as well as ion implantationtreatment [30,31], etc. have been widely used tointroduce hydrophilic groups onto the hydrophobicsynthetic biomaterials.Poly-l-lactic acid (PLLA), which has been approved

by the Food and Drug Administration for clinicalapplications, is a biodegradable polymer and has beenused to build three-dimensional scaffold for the regen-eration of tissue-engineered organs due to its biodegrad-ability, good mechanical properties and properdegradation rate [32]. However, the surface of PLLAis hydrophobic and does not posses physiological

$Financial support: The Major State Basic Research Program of

China (G1999054305).

*Corresponding author. Tel.:+86-571-87951108; fax:+86-571-

87951948.

E-mail address: cygao@mail.hz.zj.cn (C. Gao).

0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0142-9612(03)00247-3

activity, which makes it unfavorable for cell adhesion[4,33]. In a previous work we have introduced OH,COOH or CONH2 groups onto PLLA membranesurface via the photo-induced grafting copolymerizationof hydroxyethyl methacrylate (HEMA), methacrylicacid (MAA) or acrylamide (AAm), respectively [3436]. In this study, chondrocyte culture was used toexamine whether or not the modied PLLA membraneshave better cytocompatibility and to study the correla-tion between the surface chemical structures and thechondrocyte behaviors.

2. Experiments

2.1. Materials

PLLA (Mn=200,000, Mw=400,000) 1,4-dioxanesolution with a concentration of 4wt% was cast ontoa stainless-steel plate and dried under vacuum to yieldPLLA membrane with a thickness ofB0.1mm. Hydro-xyethyl methacrylate (HEMA, Sigma) and methacrylicacid (MAA, Sigma) were puried by distillation undervacuum. Acrylamide (AAm, Sigma) was puried byrecrystallization from acetone.

2.2. Grafting copolymerization of the hydrophilic vinyl

monomers onto the PLLA membranes

Hydrophilic groups were introduced onto the PLLAmembrane surface using the method described pre-viously [3436]. Briey, the PLLA membrane was placedin hydrogen peroxide solution (30%) under UV light at50C for 40min. Then the photo-oxidized membranewas rinsed with deionized water and immersed intomonomer solution (5%) in a Pyrex glass tube purgedwith nitrogen. Grafting copolymerization was carriedout under UV irradiation at 50C for 60min. Thegrafted membrane was rinsed with deionized water at70C for 24 h to remove the adsorbed homopolymers.Atomic force microscopy (AFM) images of the originaland the grafted PLLA membranes were obtained on anSPI3800N machine using taping mode. Water contactangle measurements were performed on a KRUSSDSA10-MK machine.

2.3. Cell culture

Chondrocytes were isolated from cartilage tissue ofrabbit ears (Japanese big ear white). Briey, cartilagetissue obtained from the rabbit ears was cut into smallpieces. Chondrocytes were isolated by incubating thecartilage pieces in F-12 HAMs (Hyclone) culturemedium containing 0.2% collagenase II (Sigma) at37C for 6 h under agitation. The chondrocytes werecentrifuged, resuspended in F-12 HAMs supplemented

with 20% fetal calf serum (FBS), 300mg/l glutamine,50mg/l vitamin C, 100U/ml penicillin and 100U/mlstreptomycin. The cell suspension was then seeded in11 cm tissue culture dishes (Falcon, seeding density2 104 cells/cm2) and incubated in a humidied atmo-sphere of 95% air, 5% CO2 at 37

C. After a conuentcell layer was formed (about 34 days), the cells weredetached using 0.25% trypsin in PBS and wereresuspended in the supplemented culture medium asdescribed above, and used for the experiments.

2.4. Cell adhesion and proliferation

The original and the modied PLLA membranes wereplaced on the bottom of the tissue culture plates (Costar,24 wells). Cell adhesion was determined at 24 h afterseeding 6 104 cells/cm2 on each specimens and tissueculture polystyrene (TCPS). Before harvesting theadherent cells by trypsinization, twice-gentle washingswith PBS were performed. The cells were counted usinga haemocytometer. The adhesion rate was dened as thepercentage of the cell number counted on the sample tothe cell number counted on TCPS. Cell proliferation wasdetermined in a similar way after culturing for 96 h witha seeding density of 3 104 cells/cm2 on each specimensand TCPS. The proliferation rate was dened as thepercentage of the cell number to the seeded cell number.All data are presented as the mean values of four counts.

2.5. Cell viability by MTT assay

Cell viability assay was performed after 96 h followingthe seeding (seeding density 3 104) on each specimensand TCPS bottom. A total of 200 ml MTT (3-(4,5-dimethyl) thiazol-2-yl-2,5-dimethyl tetrazolium bro-mide, 5mg/ml) was added to each culture well andincubated for 4 h. The MTT was reduced to formazanpigment by living cells. Finally, the culture medium wasremoved and dimethyl sulphoxide (DMSO) was added.The absorbance at 490 nm was measured. The relativecell viability was dened as the ratio of the absorbancefrom the sample to the TCPS.

2.6. Cell morphological assessment

After being xed with 2.5% glutaraldehyde for30min, the cells were stained with Giemsa (The ThirdChemical Co. of Shanghai, China). Microscopic ob-servation of the cell morphology was carried out usingan inverted microscope.

3. Results and discussion

It has been demonstrated that through the photo-induced grafting copolymerization, the hydrophilic

Z. Ma et al. / Biomaterials 24 (2003) 372537303726

groups can be introduced onto the PLLA membranesurface and the hydrophilicity can be improvedobviously [36]. Water contact angles of the originaland the modied PLLA membranes are listed inTable 1. The captive bubble contact angles (CBCA) ofall the specimens were lower than the correspondingsessile drop contact angles (SCA). All the modiedPLLA membranes had similar CBCA (B40), whichwere much lower than that of the original membrane(72). It should be noted that the CBCA values aremuch close to the real situation when the modiedPLLA are employed as tissue regeneration scaffoldseither in vitro or in vivo.Fig. 1 shows the AFM images of the original and the

modied PLLA membranes. No big difference betweenthe surface morphology of the original and the modiedPLLA membranes was observed. The cell adhesion,

proliferation and viability on the original and themodied PLLA membranes are shown in Fig. 2. Figs. 3and 4 presented the light micrographs of the chondro-cytes on the original and the modied membranes at 48and 96 h after seeding the cells, respectively. It can beseen from Fig. 2 that except the PLLA membranegrafted with PHEMA (PLLA-g-PHEMA) had a slightlyhigher cell adhesion and proliferation rate than theoriginal membrane, no obvious differences of celladhesion, proliferation rate and viability were detectedbetween the original and the other modied membranes.These data means that the cell numbers obtained fromthe different specimens were rather similar.However, the cells on different specimens showed

obviously different morphology. It has been reportedthat cell shape affects cell growth, gene expression,extracellular matrix metabolism and differentiation [3739]. Thus, the cell morphology is a hint of the cellcapacity for proliferation and differentiation, and can beused to determine whether the biomaterial has a goodcytocompatibility. At 48 h after seeding, the chondro-cytes were spherical and round on the original mem-brane or the membrane grafted with PMAA (PLLA-g-PMAA), whilst on the membranes grafted with PHE-MA or PAAm (PLLA-g-PAAm), the cells were at,polygonal and more spread out, which is the normalshape of chondrocyte [22]. Moreover, the cells on the

Table 1

Water contact angle of the original and the modied PLLA

membranes

SCA (deg) CBCA (deg)

Original PLLA 82.072.3 72.272.4PLLA-g-PHEMA 51.175.54 40.173.59PLLA-g-PAAm 65.474.01 41.873.76PLLA-g-PMAA 51.073.15 39.874.41

Each value was averaged from 15 measurements.

Fig. 1. Atomic force micrographs of the original and the modied PLLA membranes: (a) original PLLA; (b) PLLA-g-PHEMA; (c) PLLA-g-PAAm;

and (d) PLLA-g-PMAA.

Z. Ma et al. / Biomaterials 24 (2003) 37253730 3727

membrane grafted with PMAA were very easy toaccumulate with each other to form cell nodules andmultilayers, indicating a rather poor cytocompatibilityof the membrane. After culturing for 4 days, conuentcell layers were formed on the PLLA-g-PHEMA orPLLA-g-PAAm, whereas serious cell accumulatingoccurred both on the original membrane and on thePLLA-g-PMAA.Due to the existing of the hydrophilic groups on the

modied membrane surfaces, all the three modiedmembranes had similar hydrophilicity that is better thanthe original membrane (Table 1). However, chondro-cytes showed different response to these modied PLLAmembranes. An obvious improvement of cytocompat-ibility was observed on the OH or CONH2 existingsurfaces. The COOH existing membrane had evenworse cytocompatibility than the original membrane.Considering the similar surface morphology of all themodied PLLA membranes (Fig. 1), this result meansthat the surface chemical structure plays also animportant role on cytocompatibility as wettability. Thereason why the PMAA grafted membrane showed poorcytocompatibility is not clear, though it may be

0

50

100

150

200

1 2 3 4 5adhesion(%,relative to TCPS,24h)proliferation rate(%,4days)cell activity(%,relative to TCPS,4days)

a b c d e

Adh

esio

n, p

rolif

erat

ion

and

activ

ity o

f ch

ondr

ocyt

es

Fig. 2. Adhesion, proliferation rate and cell viability of chondrocytes

on (a) TCPS; (b) original PLLA; (c) PLLA-g-PHEMA; (d) PLLA-g-

PAAm; and (e) PLLA-g-PMAA.

(a) (b)

(c) (d)

(e)

Fig. 3. Chondrocyte morphology on (a) TCPS; (b) original PLLA; (c) PLLA-g-PHEMA; (d) PLLA-g-PAAm; and (e) PLLA-g-PMAA at 48 h after

seeding density of 40,000/cm2, Bar=100mm.

Z. Ma et al. / Biomaterials 24 (2003) 372537303728

attributed to the negative charge property [40,41].However, our previous results do have revealed thatpolycaprolactone grafted with PMAA had a bettercytocompatibility for human endothelial cells [42].Studies are underway to explore the inuence of graftingmethod and grafting amount on cell adhesion, growthand spreading behaviors.

4. Conclusions

Three kinds of hydrophilic polymers, i.e. polyhydrox-yethyl methacrylate (PHEMA), polymethacrylic acid(PMAA) or polyacrylamide (PAAm) have been linkedonto the PLLA membranes via the photo-inducedgrafting copolymerization of the corresponding mono-mers to introduce hydroxyl groups (OH), carboxylgroups (COOH) or amide groups (CONH2), respec-tively. All the modied membranes have better hydro-philicity than the original membrane. However, onlysurface containing OH or CONH2 groups have better

cytocompatibility, while COOH not. Therefore, it canbe inferred that the surface chemical structure takes animportant role as the surface wettability in improvingthe cytocompatibility for chondrocytes.

Acknowledgements

This research is nancially supported by The MajorState Basic Research Program of China (G1999054305).

References

[1] Canans M, Denicolai F, Webb LX, Gristina AG. Bioimplant

surfaces: binding of bronectin and broblast adhesion. J Orthop

Res 1988;6:5862.

[2] Perez-Luna VH, Horbert TA, Ratner BD. Developing correla-

tions between brinogen adsorption and surface properties using

multivariate statistics. J Biomed Mater Res 1994;28:111126.

[3] Uyen HM, Schakenraad JM, Sjollema J. Amount and surface

structure of albumin adsorbed to solid substrata with different

(a) (b)

(c) (d)

(e)

Fig. 4. Chondrocyte morphology on (a) TCPS; (b) original PLLA; (c) PLLA-g-PHEMA; (d) PLLA-g-PAAm; and (e) PLLA-g-PMAA at 96 h after

seeding density of 40,000/cm2, Bar=100mm.

Z. Ma et al. / Biomaterials 24 (2003) 37253730 3729

wettabilities in a parallel plate ow cell. J Biomed Mater Res

1990;24:1599614.

[4] van Wachem PB, Beugelling T, Feijen J, Bantjes A, Detmers JP,

van Aken WG. Interaction of cultured human endothelial cells

with polymeric surfaces of different wettabilities. Biomaterials

1985;6:403.

[5] Clark P, Moores GR. Cell guidance by micropatterned adhesive-

ness in vitro. J Cell Sci 1992;103:287.

[6] van Wachem PB, Hoget AH, Beugeling T, Feijen J, Bantjes A,

Detmers JP, van Aken WG. Adhesion of cultured human

endothelial cells onto methacrylate polymers with varying surface

wettability and charge. Biomaterials 1987;8:323.

[7] Khang G, Lee SJ, Jeon JH, Lee JH, Lee HB. Interaction of

broblast cell onto physicochemically treated PLGA surfaces.

Polymer-Korea 2000; 24: 86976.

[8] Lee JH, Lee SK, Khang G, Lee HB. The effect of uid sheer stress

on endothelial cell adhesiveness to polymer surfaces with

wettability gradient. J Colloid Interface Sci 2000;230:8490.

[9] Khang G, Lee SJ, Lee YM, Lee JH, Lee HB. The effect of

uid sheer stress on endothelial cell adhesiveness to modied

polyurethane surfaces. Korea Polym J 2000;8:17985.

[10] Davies JE, Causton B, Bovell Y, Davy K. The migration of

osteoblasts over substrata of discrete surface charge. Biomaterials

1986;7:2313.

[11] Kornu R, Maloney WJ, Kelly MA. Osteoblast adhesion to

orthopaedic implant alloys: effects of cell adhesion molecules and

diamond-like carbon coating. J Orthop Res 1996;14:8717.

[12] Sinha RK, Morris F, Shah SA, Tuan RS. Surface composition of

orthopaedic implant metals regulates cell attachment, spreading,

and cytoskeletal organization of primary human osteoblasts in

vitro. Clin Orthop Rel Res 1994;305:25872.

[13] Sinha RK, Shah SA, Tuan RS. Cell spreading and attachment of

human osteoblasts to metallic substrates. J Cell Biol 1991;115:

448a.

[14] Schakenraad JM, Busscher HJ, Wildevuur CR. The inuence of

substratum free energy on growth and spreading of human

broblasts in the presence and absence of serum proteins.

J Biomed Mater Res 1986;20:77384.

[15] Curtis A. Cell activation and adhesion. J Cell Sci 1987;87:60911.

[16] Horbett TA, Schway MB, Ratner BD. Hydrophilichydrophobic

copolymers as cell substrates: effect on 3T3 cell growth rate.

J Colloid Interface Sci 1985;104:2839.

[17] Horbett TA, Schway MB. Correlations between mouse 3T3 cell

spreading and serum bronectin adsorption on glass and

hydroxyethylmethacrylate-ethylmethacrylate polymers. J Biomed

Mater Res 1988;22:76393.

[18] Chinn JA, Horbett TA, Ratner BD, SchwayMB. Enhancement of

serum bronectin adsorption and the clonal plating efciencies of

Swiss mouse 3T3 broblast and MM14 mouse myoblast cells on

polymer substrates modied by radiofrequency plasma deposi-

tion. J Colloid Interface Sci 1989;127:6787.

[19] Ertel SI, Ratner BD, Horbett TA. Radiofrequency plasma

deposition of oxygen-containing lms on polystyrene and poly

(ethyleneterephthalate) substrates improves endothelial cell

growth. J Biomed Mater Res 1990;24:163759.

[20] Evans MDM, Steele JG. Polymer surface chemistry and a novel

attachment mechanism in corneal epithelial cells. J Biomed Mater

Res 1998;40:62130.

[21] Dahm M, Chang BJ, Prucker O. Surface attached ultrathin

polymer monolayers for control of cell adhesion. Ann Thorac

Surg 2001;71:S43740.

[22] Ishaug-Riley SL, Okun LE, Prado G, Applegate MA. Human

articular chondrocyte adhesion and proliferation on synthetic

biodegradable polymer lms. Biomaterials 1999;20:224556.

[23] Steele JG, John G, MeLean KM, Beumer GJ, Griesser HJ. Effect

of porosity and surface hydrophilicity on migration of epithelial

tissue over synthetic polymer. J Biomed Mater Res 2000;50:

47582.

[24] Muller M, Oehr C. Plasma aminofunctionalisation of PVDF

microltration membranes: comparison of the in plasma mod-

ications with a grafting method using ESCA and an amino-

selective uorescent probe. Surf Coatings Technol 1999;

116119:8027.

[25] Klee D, Villari RV, Hocker H, Dekker B, Mittermayer C. Surface

modication of a new exible polymer with improved cell

adhesion. J Mater Sci: Mater Med 1995;5:5925.

[26] Fang YE, Lu XB, Wang SZ, Zhao X, Fang F. Study of radiation-

induced graft copolymerization of vinyl acetate onto ethylene-co-

propylene rubber. J Appl Polym Sci 1996;62:220913.

[27] Ajayaghosh A, Das S. Photografting of acrylic monomers on

polystyrene support. J Appl Polym Sci 1992;45:161722.

[28] Geuskens G, Etoc A, Michele PD. Surface modication of

polymers VII. Photochemical grafting of acrylamide and N-

isopropylacrylamide onto polyethylene initiated by anthraqui-

none-2-sulfonate adsorbed at the surface of the polymer. Eur

Polym J 2000;36:26571.

[29] Kang IK, Kwon BK, Lee JH, Lee HB. Immobilization of

proteins on poly(methyl methacrylate) lms. Biomaterials 1993;

14:78791.

[30] Lee JS, Kaibara M, Iwaki M, Sasabe H, Suzuki Y, Kusakabe M.

Selective adhesion and proliferation of cells on ion-implanted

polymer domains. Biomaterials 1993;14:95860.

[31] Sato H, Tsuji H, Ikeda S, Ikemoto N, Ishikawa J, Nishimoto S.

Enhanced growth of human vascular endothelial cells on negative

ion (Ag)- implanted hydrophobic surfaces. J BiomedMater Res.1999;44:2230.

[32] Freed LE, Marquis JC, Nohria A, Emmanuel J, Mikos AG,

Langer R. Neocartilage formation in vitro and in vivo using cells

cultured on synthetic biodegradable polymers. J Biomed Mater

Res 1993;27:1123.

[33] Mikos AG, Lyman MD, Freed LE, Langer R. Wetting of

poly (l-lactic acid) and poly (dl-lactic-co-glycolic acid) foams fortissue culture. Biomaterials 1994;15:558.

[34] Feng XD, Sun YH, Qiu KY. Selective grafting of hydrogels onto

multiphase block copolymers. Makromol Chem 1985;186:1533.

[35] Guan JJ, Gao CY, Feng LX, Shen JC. Functionalizing of

polyurethane surfaces by photografting with hydrophilic mono-

mers. J Appl Polym Sci 2000;77:250512.

[36] Ma ZW, Gao CY, Yuan J, Ji J, Gong YH, Shen JC. Surface

modication of poly l-lactide by photografting of hydrophilicpolymers towards improving its hydrophilicity. J Appl Poly Sci

2002;85:216371.

[37] Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE.

Science 1997;276:1425.

[38] Singhvi R, Kumar A, Lopez GP, Stephanopoulos GN, Wang

DIC, Whitesides GM, Ingber DE. Eng Cell Shape Function Sci

1994;264:6968.

[39] Huang S, Ingber DE. Nat Cell Biol 1999:E131.

[40] Lee JH, Khang G, Lee JW, Lee HB. Platelet adhesion onto

chargeable functional group gradient surfaces. J Biomed Mater

Res 1998;40:1806.

[41] Lee JH, Lee JW, Khang G, Lee HB. Interaction of cells on

chargeable functional group gradient surfaces. Biomaterials

1997;18:3518.

[42] Zhu YB, Gao CY, Shen JC. Surface modication of polycapro-

lactone with poly(methacrylic acid) and gelatin covalent immo-

bilization for promoting its cytocompatibility. Biomaterials

2002;23:488995.

Z. Ma et al. / Biomaterials 24 (2003) 372537303730

Chondrocyte behaviors on poly-l-lactic acid (PLLA) membranes containing hydroxyl, amide or carboxyl groupsIntroductionExperimentsMaterialsGrafting copolymerization of the hydrophilic vinyl monomers onto the PLLA membranesCell cultureCell adhesion and proliferationCell viability by MTT assayCell morphological assessment

Results and discussionConclusionsAcknowledgementsReferences