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Fibroblast culture on surface-modified poly (glycolide-co-ε-caprolactone) scaffold for softtissue regenerationI K Kwon a K D Park b S W Choi c S-H Leed E B Lee e J S Na f S H Kim g amp YH Kim ha Biomaterials Research Center Korea Instituteof Science and Technology PO Box 131Cheongryang Seoul 130-650 Koreab Department of Molecular Science andTechnology Ajou University Suwon Kyungkido442-749 Koreac Biomaterials Research Center Korea Instituteof Science and Technology PO Box 131Cheongryang Seoul 130-650 Koread Biomaterials Research Center Korea Instituteof Science and Technology PO Box 131Cheongryang Seoul 130-650 Koreae Biomedical Research Center Korea Instituteof Science and Technology PO Box 131Cheongryang Seoul 130-650 Koreaf Department of Chemical Engineering KwangwoonUniversity Seoul 139-701 Koreag Biomaterials Research Center Korea Instituteof Science and Technology PO Box 131Cheongryang Seoul 130-650 Koreah Biomaterials Research Center Korea Instituteof Science and Technology PO Box 131Cheongryang Seoul 130-650 Korea

Published online 02 Apr 2012

To cite this article I K Kwon K D Park S W Choi S-H Lee E B Lee J S Na S H Kim amp YH Kim (2001) Fibroblast culture on surface-modifiedpoly (glycolide-co-ε-caprolactone) scaffold for soft tissue regeneration Journal of Biomaterials Science Polymer Edition 1210 1147-1160 DOI10116315685620152691904

To link to this article httpdxdoiorg10116315685620152691904

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J Biomater Sci Polymer Edn Vol 12 No 10 pp 1147ndash1160 (2001)Oacute VSP 2001

Fibroblast culture on surface-modi ed poly(glycolide-co-e -caprolactone) scaffold for soft tissueregeneration

I K KWON 1 K D PARK 2 S W CHOI 1 S-H LEE 1 E B LEE 3 J S NA 4S H KIM 1 and Y H KIM 1curren

1 Biomaterials Research Center Korea Institute of Science and Technology PO Box 131Cheongryang Seoul 130-650 Korea

2 Department of Molecular Science and Technology Ajou University SuwonKyungkido 442-749 Korea

3 Biomedical Research Center Korea Institute of Science and Technology PO Box 131Cheongryang Seoul 130-650 Korea

4 Department of Chemical Engineering Kwangwoon University Seoul 139-701 Korea

Received 10 October 2000 revised 4 June 2001 accepted 31 July 2001

AbstractmdashNovel porous matrices made of a copolymer of glycolide (G) and -caprolactone (CL)(51 49 Mw 103 000) was prepared for tissue engineering using a solvent-castingparticulate leachingmethod Poly(glycolide-co--caprolactone) (PGCL) copolymer showed a rubber-like elastic charac-teristic in addition to an amorphous property and fast biodegradability In order to investigate the ef-fect on the broblast culture PGCL scaffolds of varying porosity and pore size in addition to surface-hydrolysis or collagen coating were studied The large pore-sized scaffold (pore size gt150 sup1m)demonstrated a much greater cell adhesion and proliferation than the small pore-sized one In addi-tion the higher porosity the better the cell adhesion and proliferation The surface-hydrolyzedPGCLscaffold showed enhanced cell adhesion and proliferation compared with the unmodi ed one TypeI collagen coating revealed a more pronounced contribution for increased cell interactions than thesurface-hydrolyzedone These results demonstrate that surface-modied PGCL scaffold can providea suitable substrate for broblast culture especially in the case of soft tissue regenerations

Key words Poly(glycolide-co--caprolactone) scaffold broblast (NIH3T3) surface modi cation

INTRODUCTION

Porous absorbable matrices made of natural or synthetic polymers are investigatedas scaffolds for the purpose of cell and tissue transplantation [1ndash4] For these

currenTo whom correspondence should be addressed E-mail yhakimkistrekr

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1148 I K Kwon et al

scaffolds proper biocompatability degradability mechanical stability high surfacearea volume ratio and interconnection are required These biodegradable polymersinclude polyglycolide (PGA) [5 6] polylactide (PLA) [4 7] poly(glycolide-co-lactide) (PGLA) poly(-caprolactone) (PCL) [8 9] collagen [10ndash12] alginate [1314] hyaluronate [15] and laminin [16] etc PGA is now used clinically as amaterial for sutures and tissue regeneration but is too strong and rigid for certainapplications In order to modulate the mechanical properties of PGA suitablecomonomers such as lactide or -caprolactone were incorporated to yield a variedfamily of bioabsorbable materials with soft and exible compositions [17] Therewere many studies carried out on the degradation mechanism and toxicology of PCLand its copolymers that were degraded into glycolic acid lactic acid and -hydroxyhexanoic acid upon hydrolysis [9 18ndash21]

The success of any cell transplantation therapy relies on the development ofsuitable substrates for both in vitro and in vivo tissue culture For ideal tissueengineering it is most important to obtain a suf cient mass of seeded cells and theiruniform distribution throughout the whole scaffold As these degradable polymersare hydrophobic in general cell suspensions (or even culture media) do not wetporous devices and penetrate into the inside so that the majority of pores remainempty are not utilized for cell culture [22] Type I collagen is frequently used asa substrate for cell culture because it has cell binding domain sequences such asextracellular matrix (ECM) molecules [22 23] The ECM molecules are generallyhydrophilic proteins and oligo- and polysaccharides [24]

The objectives of this research are to prepare proper structural PGCL (50 50)scaffolds by a solvent-casting particulate leaching method [25] and to modify thescaffold surfaces by hydrolysis [26] or collagen coating for the investigation ofenhanced initial adhesion and broblast proliferation [2 12]

MATERIALS AND METHODS

Materials

Glycolide (Boehringer Ingelheim) was puri ed by recrystallization from dry toluene-Caprolactone (Aldrich Chemical) was puri ed by drying over CaH2 and distillingunder reduced nitrogen atmosphere The catalyst stannous octoate (Sigma Chemi-cal) was used as-received Unless otherwise speci ed all the chemicals were pur-chased from Sigma Chemical

Synthesis of poly(glycolide-co--caprolactone) (PGCL)

A copolymer of glycolide 5707 g (0500 mol) and -caprolactone 5804 g(0500 mol) was prepared in silanized glass ampoules To the mixture of freshlypuri ed monomers an amount of 00150 g (130 pound 10iexcl5 mol) catalyst was addedafter which the ampoules were evacuated and heat-sealed in nitrogen atmosphere

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Fibroblast culture on surface-modied PGCL 1149

The polymerization reaction was carried at 170plusmnC in a silicone oil bath for 20 hThe copolymer was recovered by dissolving in CHCl3 and puri ed by precipitationin methanol (Fisher Co) For comparison a copolymer of glycolide and L-lactide(70 30 molmol) was copolymerized by the analogous method

Characterizations

The prepared PGCL copolymer was analyzed by 600 MHz 1H-NMR (VarianFT-NMR spectrometer) Hexa uoroacetone deuterate was used as solvent andtetramethyl silane (TMS) as an internal standard NMR spectra were obtained at7plusmnC PGCL (0072 g) was dissolved in 144 ml hexa uoroisopropanol (HFIP) andmeasured for inherent viscosity by an Ubbelohde viscometer The molecular weightof the PGCL copolymer was determined by gel permeable chromatography (GPCModel 510 pump equipped with a 410 Differential Refractometer) using threemicro-styragel columns (CHR2 HR4 and HR5E) from 500 to 4 pound 106Aring connectedin series Tetrahydrofuran (THF) was eluted at a rate of 10 ml miniexcl1 at 40plusmnCCalibration was carried out using polystyrene standards (Shodex) over a molecularweight range of 130 pound 103 ndash 196 pound 106

Thermal analysis of the polymer was carried out on a differential scanningcalorimeter (DSC Du Pont TA 2000) employing a heating rate of 10plusmnC miniexcl1Tensile property was measured by Instron (Model 5567) Samples were cut into1 pound 1 cm for compression and tensile testing The specimens attached to cardboardusing epoxy glue were drawn at a constant rate (1 mm miniexcl1 )

Static water contact angles on PGCL surfaces were measured by the sessiledrop method using deionized distilled water by a goniometer (model G-1 ErmaInc) Chemical structural changes on the hydrolyzed surface of the sampleswere examined by electron spectra for chemical analysis (ESCA Surface ScienceInstrument model 2803-S)

Preparation of PGCL scaffolds

PGCL porous membranes were prepared by a solvent-casting particulate leachingtechnique [25] that consisted of the following steps

(1) The sieved NaCl particles were added into 5 wtwt PGCL solutions inchloroform and the vortexed dispersions were cast in glass Petri dishes (diameter5 cm) Five different polymer salt compositions were applied in this study 30702080 1090 595 and 397 by wt ratios while the salt size was xed at150 lt d lt 300 sup1m For the 595 wt ratio four different salt sizes were used0 lt d lt 53 53 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m (2) Thesolvent was allowed to evaporate from the covered Petri dish at room temperatureover 48 h and then completely by vacuum drying at room temperature for 24 h(3) The PGCLsalt composite membranes were immersed in distilled deionizedwater with stirring for 48 h (the water was changed at every 12 h) to leach out the

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1150 I K Kwon et al

salt (4) The salt-free PGCL membranes were air-dried for 24 h and then vacuum-dried for 48 h The scaffolds were stored in a desiccator under vacuum

Degradation study of PGCL scaffolds

The degradation study of PGCL scaffolds was conducted in phosphate-bufferedsaline (PBS) at pH 74 at 37plusmnC using a shaking incubator (100 rpm) The pH of themedium was continuously monitored and the medium was Freshly replaced every48 h Weight loss and molecular weights were measured by GPC at certain timeperiods

Surface modi cation

For the hydrolysis the lms and scaffolds were placed in 1 N NaOH solution withshaking for 1 3 5 10 and 20 min respectively They were rinsed with waterextensively until the rinsing water became neutral nally washed with ethanol andfreeze-dried The elemental compositions of carbon and oxygen on the surfaceof the PGCL lms were obtained by ESCA Collagen (Type I Sigma C3929)was dissolved in 0001 N HCl solution to 01 wt concentration The scaffoldswere immersed in 70 ethanol for 5 min and then into the collagen solutions for30 min [28] and dried under vacuum for 24 h In order to decrease the degradationrate of the collagen by cross-linking the coated scaffolds were irradiated by UV254 nm at 4plusmnC for 2 h The specimens were exposed to eight surrounding 10-W UVlamps (Daeil DBO231S) and the distance between the light source and sample was5 in [28 37] Finally they were washed with PBS until the rinsing water becameneutral

Determination of the amount of type I collagen coated

The amount of collagen coated was assayed by the Bradford method [29] Inaddition it was con rmed whether the collagen coated was practically cross-linkedby UV irradiation The polymer sponges were incubated in Dulbeccorsquos modi edEaglersquos medium (DMEM Sigma) without fetal bovine serum (FBS) for 0 and 24 hin a CO2 incubator After incubation the polymer sponge was placed in 6 N HClsolution at 120plusmnC while the coated collagen was separated from the sponge anddissolved into the solution The absorbance of the solution was measured at 610 nmby an enzyme-linked immunosorbent assay (ELISA) apparatus (Spectra Max 340Molecular Device Inc CA USA)

Cell culture studies

Fibroblasts (NIH3T3 ATCC)were harvested after trypsin treatment (005 trypsin 002 EDTA Gibco) and cultured in DMEM supplemented with 10 bovine calfserum and 50 U mliexcl1 of penicillin and streptomycin (Sigma) The cells were main-tained at 37plusmnC in humidi ed 5 CO2 atmosphere All the data were presented as

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Fibroblast culture on surface-modied PGCL 1151

the mean values of three counts All the scaffolds were sterilized by UV irradia-tion for 5 min and immersed in 70 ethanol overnight and then washed with PBSthree times Cell suspensions (50 sup1l) containing a certain number of cells (5 pound 105

cells mliexcl1 for the porosity study 1 pound 106 cells mliexcl1 for the pore size study and5 pound 106 cells mliexcl1 for the surface modi cation study) were seeded onto polymerscaffolds (5 pound 5 pound 12 mm) located in 24-well tissue culture plates After 3 h 1 mlof culture medium was added to each well Cell adhesion and proliferation on thescaffolds were determined at 1 and 3 days after seeding The matrices were rinsedwith PBS to remove unattached cells before harvesting the adherent cells and cellcounting Cell numbers were determined by a uorometric quanti cation of DNAin the scaffolds by an assay according to West et al [30] An aliquot was read u-orometrically (Hoefer Scienti c Instruments DNA Fluorometer) using Hoechst dyeno 33258 (Polyscience Inc) Fluorescence emissions at 455 nm of the supernatantsamples were read with the excitation set at 350 nm The amount of DNA wasextrapolated from the standard curve of broblast (NIH3T3) DNA

Fibroblasts seeded onto PGCL scaffolds were xed for examination by scanningelectron microscope (SEM Stereoscan 360 Cambridge Instruments UK) Thescaffolds were washed with PBS and then xed in PBS containing 2 glutaldehydeat room temperature for 1 h followed by a incubation for 24 h at 4plusmnC After washingagain with PBS buffer the scaffolds were dehydrated through a graded series of 5060 70 80 90 and 100 ethanol each time for 10 min The samples were dried andcoated with an ultra-thin gold layer (100 Aring)

Statistical analysis

All the quantitative results were obtained from triplicate samples Data wereexpressed as a mean sect SD Statistical analysis was carried out using the unpairedStudentrsquos t-test A value of p lt 005 was considered to be statistically signi cant

RESULTS

Characterization of poly(glycolide-co--caprolactone) (PGCL)

The copolymer was characterized in terms of the thermal properties and microstruc-ture by means of viscosimetry DSC NMR spectroscopy and GPC Table 1 showsthat the PGCL had a molar composition (G CL) of 51 49 by NMR The mole-cular weight (Mw ) was measured at 103 000 by GPC and the intrinsic viscositywas 093 dl giexcl1 in chloroform The glass transition temperature (Tg ) was observedat iexcl154plusmnC but no crystallization temperature (Tc) or melting point (Tm ) to indi-cate the PGCL synthesized to be random and amorphous The PGCL revealed amuch lower tensile modulus but higher elongation than PGLA (70 30) (Mw wasabout 300 000) did as shown in Fig 1a The PGCL prepared in this study wasrubber-like elastic to show an extension over 250 and a full recovery as observed

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1152 I K Kwon et al

Table 1Characteristics of PGCL copolymer prepared

Mole ratio Composition a Yield Tgb Tc

b Tmb Mw

c acuteinhd

in feed of copolymer () ( plusmnC) ( plusmnC) ( plusmnC) (g dliexcl1)[G][CL] [G][CL]

5050 5149 962 iexcl154 mdash mdash 103 000 093

a Measured by 1H-NMRb Measured by DSC at a heating rate of 10plusmnC miniexcl1c Measured by GPC in chloroform at 1 ml miniexcl1 30plusmnCd Inherent viscosity in HFIP at 05 g dliexcl1 at 25plusmnC

(a)

(b)

Figure 1 Mechanical strength of PGCL (51 49) and PGLA (70 30) scaffolds (a) stressndashstraincurve of PGCL and PGLA and (b) elastic recovery of PGCL

in Fig 1b Porous PGCL matrices were prepared by a solvent-casting particulateleaching method using sodium chloride particles

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Fibroblast culture on surface-modied PGCL 1153

Preparation of PGCL scaffolds

The effects of the weight fraction and particle size of sodium chloride on theporosity pore diameter and surface volume ratio were investigated First of all thematrix porosity increased with increasing the weight fraction of sodium chlorideThe mean pore diameter increased too as the salt particle size increased The poresize was evaluated by a boundary area calculation method from a SEM picture [31]The mean pore sizes were 22 70 150 and 300 sup1m respectively for particle sizes of0 lt d lt 50 50 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m Membranesprepared with salt weight fractions above 90 wt had a uniform pore distribution asobserved by SEM however those with 70 or 80 wt formed asymmetric structureswith a dense impermeable skin at the bottom surface of the scaffolds

Degradation study of PGCL scaffolds

The degradation study of PGCL lm and scaffold (made with 95 wt salt poresize 150 sup1m) was conducted in PBS at 37plusmnC with a shaking incubator (100 rpm)The weight loss and molecular weight by GPC were measured as shown in Fig 2The weight of PGCL lm did not change up to 2 weeks but decreased graduallyand rapidly after 3 weeks At 6 weeks 60 of the initial mass was degraded Themolecular weights of PGCL lm and scaffold were rapidly reduced throughout thewhole period as shown in Fig 2b The smaller initial molecular weight of thescaffold than that of the lm may be caused by partial hydrolysis at the immersingprocess in water during scaffold preparation This result indicated that the PGCLcopolymer would be degraded mostly in 8ndash10 weeks to leave low Mw fractions

Surface hydrolysis and determination of the density of collagen coated

To enhance the hydrophilicity of the surface by introducing free carboxylic andhydroxyl end groups the PGCL scaffold was partially hydrolyzed by soaking inaqueous 1 N NaOH solution In addition the PGCL lm was treated under thesame conditions for ESCA and contact angle study Figure 3 shows the surfacemorphology of the PGCL scaffolds unmodi ed and hydrolyzed for 1 3 5 10 and20 min respectively It was revealed that the hydrolysis caused surface erosionresulting in increased pore size In addition the wettability of the PGCL wassigni cantly increased on hydrolysis The static contact angles of the unmodi edPGCL lm and the surface-hydrolyzed ones were measured by sessile drop methodas shown in Fig 4 The contact angle was gradually reduced with increasingtime to indicate approximately 65 deg after 3 min which was a substantiallydecreased value from 84 deg for the untreated one The changes of the surfaceelemental compositions of the hydrolyzed lms were analyzed by ESCA Thesurface composition of unmodi ed PGCL lm indicated 76 carbon and 24oxygen After hydrolysis they changed to 73 carbon and 27 oxygen as shownin Table 2 The increase in oxygen content on the surface-hydrolyzed PGCL lmshould be a result of the carboxylic and hydroxy groups evolved by the hydrolysis

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1154 I K Kwon et al

(a)

(b)

Figure 2 Degradation behavior of PGCL (a) weight loss of lm and (b) molecular weight loss ofscaffold (-pound-) and lm (-F-)

Table 2Atomic compositionof PGCL by ESCA analysisafter hydrolyzation for 3 min

Samples C O OC

Untreated PGCL 7628 2372 031Hydrolyzed PGCL 7280 2720 037

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Fibroblast culture on surface-modied PGCL 1155

Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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1156 I K Kwon et al

(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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1158 I K Kwon et al

Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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1148 I K Kwon et al

scaffolds proper biocompatability degradability mechanical stability high surfacearea volume ratio and interconnection are required These biodegradable polymersinclude polyglycolide (PGA) [5 6] polylactide (PLA) [4 7] poly(glycolide-co-lactide) (PGLA) poly(-caprolactone) (PCL) [8 9] collagen [10ndash12] alginate [1314] hyaluronate [15] and laminin [16] etc PGA is now used clinically as amaterial for sutures and tissue regeneration but is too strong and rigid for certainapplications In order to modulate the mechanical properties of PGA suitablecomonomers such as lactide or -caprolactone were incorporated to yield a variedfamily of bioabsorbable materials with soft and exible compositions [17] Therewere many studies carried out on the degradation mechanism and toxicology of PCLand its copolymers that were degraded into glycolic acid lactic acid and -hydroxyhexanoic acid upon hydrolysis [9 18ndash21]

The success of any cell transplantation therapy relies on the development ofsuitable substrates for both in vitro and in vivo tissue culture For ideal tissueengineering it is most important to obtain a suf cient mass of seeded cells and theiruniform distribution throughout the whole scaffold As these degradable polymersare hydrophobic in general cell suspensions (or even culture media) do not wetporous devices and penetrate into the inside so that the majority of pores remainempty are not utilized for cell culture [22] Type I collagen is frequently used asa substrate for cell culture because it has cell binding domain sequences such asextracellular matrix (ECM) molecules [22 23] The ECM molecules are generallyhydrophilic proteins and oligo- and polysaccharides [24]

The objectives of this research are to prepare proper structural PGCL (50 50)scaffolds by a solvent-casting particulate leaching method [25] and to modify thescaffold surfaces by hydrolysis [26] or collagen coating for the investigation ofenhanced initial adhesion and broblast proliferation [2 12]

MATERIALS AND METHODS

Materials

Glycolide (Boehringer Ingelheim) was puri ed by recrystallization from dry toluene-Caprolactone (Aldrich Chemical) was puri ed by drying over CaH2 and distillingunder reduced nitrogen atmosphere The catalyst stannous octoate (Sigma Chemi-cal) was used as-received Unless otherwise speci ed all the chemicals were pur-chased from Sigma Chemical

Synthesis of poly(glycolide-co--caprolactone) (PGCL)

A copolymer of glycolide 5707 g (0500 mol) and -caprolactone 5804 g(0500 mol) was prepared in silanized glass ampoules To the mixture of freshlypuri ed monomers an amount of 00150 g (130 pound 10iexcl5 mol) catalyst was addedafter which the ampoules were evacuated and heat-sealed in nitrogen atmosphere

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Fibroblast culture on surface-modied PGCL 1149

The polymerization reaction was carried at 170plusmnC in a silicone oil bath for 20 hThe copolymer was recovered by dissolving in CHCl3 and puri ed by precipitationin methanol (Fisher Co) For comparison a copolymer of glycolide and L-lactide(70 30 molmol) was copolymerized by the analogous method

Characterizations

The prepared PGCL copolymer was analyzed by 600 MHz 1H-NMR (VarianFT-NMR spectrometer) Hexa uoroacetone deuterate was used as solvent andtetramethyl silane (TMS) as an internal standard NMR spectra were obtained at7plusmnC PGCL (0072 g) was dissolved in 144 ml hexa uoroisopropanol (HFIP) andmeasured for inherent viscosity by an Ubbelohde viscometer The molecular weightof the PGCL copolymer was determined by gel permeable chromatography (GPCModel 510 pump equipped with a 410 Differential Refractometer) using threemicro-styragel columns (CHR2 HR4 and HR5E) from 500 to 4 pound 106Aring connectedin series Tetrahydrofuran (THF) was eluted at a rate of 10 ml miniexcl1 at 40plusmnCCalibration was carried out using polystyrene standards (Shodex) over a molecularweight range of 130 pound 103 ndash 196 pound 106

Thermal analysis of the polymer was carried out on a differential scanningcalorimeter (DSC Du Pont TA 2000) employing a heating rate of 10plusmnC miniexcl1Tensile property was measured by Instron (Model 5567) Samples were cut into1 pound 1 cm for compression and tensile testing The specimens attached to cardboardusing epoxy glue were drawn at a constant rate (1 mm miniexcl1 )

Static water contact angles on PGCL surfaces were measured by the sessiledrop method using deionized distilled water by a goniometer (model G-1 ErmaInc) Chemical structural changes on the hydrolyzed surface of the sampleswere examined by electron spectra for chemical analysis (ESCA Surface ScienceInstrument model 2803-S)

Preparation of PGCL scaffolds

PGCL porous membranes were prepared by a solvent-casting particulate leachingtechnique [25] that consisted of the following steps

(1) The sieved NaCl particles were added into 5 wtwt PGCL solutions inchloroform and the vortexed dispersions were cast in glass Petri dishes (diameter5 cm) Five different polymer salt compositions were applied in this study 30702080 1090 595 and 397 by wt ratios while the salt size was xed at150 lt d lt 300 sup1m For the 595 wt ratio four different salt sizes were used0 lt d lt 53 53 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m (2) Thesolvent was allowed to evaporate from the covered Petri dish at room temperatureover 48 h and then completely by vacuum drying at room temperature for 24 h(3) The PGCLsalt composite membranes were immersed in distilled deionizedwater with stirring for 48 h (the water was changed at every 12 h) to leach out the

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1150 I K Kwon et al

salt (4) The salt-free PGCL membranes were air-dried for 24 h and then vacuum-dried for 48 h The scaffolds were stored in a desiccator under vacuum

Degradation study of PGCL scaffolds

The degradation study of PGCL scaffolds was conducted in phosphate-bufferedsaline (PBS) at pH 74 at 37plusmnC using a shaking incubator (100 rpm) The pH of themedium was continuously monitored and the medium was Freshly replaced every48 h Weight loss and molecular weights were measured by GPC at certain timeperiods

Surface modi cation

For the hydrolysis the lms and scaffolds were placed in 1 N NaOH solution withshaking for 1 3 5 10 and 20 min respectively They were rinsed with waterextensively until the rinsing water became neutral nally washed with ethanol andfreeze-dried The elemental compositions of carbon and oxygen on the surfaceof the PGCL lms were obtained by ESCA Collagen (Type I Sigma C3929)was dissolved in 0001 N HCl solution to 01 wt concentration The scaffoldswere immersed in 70 ethanol for 5 min and then into the collagen solutions for30 min [28] and dried under vacuum for 24 h In order to decrease the degradationrate of the collagen by cross-linking the coated scaffolds were irradiated by UV254 nm at 4plusmnC for 2 h The specimens were exposed to eight surrounding 10-W UVlamps (Daeil DBO231S) and the distance between the light source and sample was5 in [28 37] Finally they were washed with PBS until the rinsing water becameneutral

Determination of the amount of type I collagen coated

The amount of collagen coated was assayed by the Bradford method [29] Inaddition it was con rmed whether the collagen coated was practically cross-linkedby UV irradiation The polymer sponges were incubated in Dulbeccorsquos modi edEaglersquos medium (DMEM Sigma) without fetal bovine serum (FBS) for 0 and 24 hin a CO2 incubator After incubation the polymer sponge was placed in 6 N HClsolution at 120plusmnC while the coated collagen was separated from the sponge anddissolved into the solution The absorbance of the solution was measured at 610 nmby an enzyme-linked immunosorbent assay (ELISA) apparatus (Spectra Max 340Molecular Device Inc CA USA)

Cell culture studies

Fibroblasts (NIH3T3 ATCC)were harvested after trypsin treatment (005 trypsin 002 EDTA Gibco) and cultured in DMEM supplemented with 10 bovine calfserum and 50 U mliexcl1 of penicillin and streptomycin (Sigma) The cells were main-tained at 37plusmnC in humidi ed 5 CO2 atmosphere All the data were presented as

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Fibroblast culture on surface-modied PGCL 1151

the mean values of three counts All the scaffolds were sterilized by UV irradia-tion for 5 min and immersed in 70 ethanol overnight and then washed with PBSthree times Cell suspensions (50 sup1l) containing a certain number of cells (5 pound 105

cells mliexcl1 for the porosity study 1 pound 106 cells mliexcl1 for the pore size study and5 pound 106 cells mliexcl1 for the surface modi cation study) were seeded onto polymerscaffolds (5 pound 5 pound 12 mm) located in 24-well tissue culture plates After 3 h 1 mlof culture medium was added to each well Cell adhesion and proliferation on thescaffolds were determined at 1 and 3 days after seeding The matrices were rinsedwith PBS to remove unattached cells before harvesting the adherent cells and cellcounting Cell numbers were determined by a uorometric quanti cation of DNAin the scaffolds by an assay according to West et al [30] An aliquot was read u-orometrically (Hoefer Scienti c Instruments DNA Fluorometer) using Hoechst dyeno 33258 (Polyscience Inc) Fluorescence emissions at 455 nm of the supernatantsamples were read with the excitation set at 350 nm The amount of DNA wasextrapolated from the standard curve of broblast (NIH3T3) DNA

Fibroblasts seeded onto PGCL scaffolds were xed for examination by scanningelectron microscope (SEM Stereoscan 360 Cambridge Instruments UK) Thescaffolds were washed with PBS and then xed in PBS containing 2 glutaldehydeat room temperature for 1 h followed by a incubation for 24 h at 4plusmnC After washingagain with PBS buffer the scaffolds were dehydrated through a graded series of 5060 70 80 90 and 100 ethanol each time for 10 min The samples were dried andcoated with an ultra-thin gold layer (100 Aring)

Statistical analysis

All the quantitative results were obtained from triplicate samples Data wereexpressed as a mean sect SD Statistical analysis was carried out using the unpairedStudentrsquos t-test A value of p lt 005 was considered to be statistically signi cant

RESULTS

Characterization of poly(glycolide-co--caprolactone) (PGCL)

The copolymer was characterized in terms of the thermal properties and microstruc-ture by means of viscosimetry DSC NMR spectroscopy and GPC Table 1 showsthat the PGCL had a molar composition (G CL) of 51 49 by NMR The mole-cular weight (Mw ) was measured at 103 000 by GPC and the intrinsic viscositywas 093 dl giexcl1 in chloroform The glass transition temperature (Tg ) was observedat iexcl154plusmnC but no crystallization temperature (Tc) or melting point (Tm ) to indi-cate the PGCL synthesized to be random and amorphous The PGCL revealed amuch lower tensile modulus but higher elongation than PGLA (70 30) (Mw wasabout 300 000) did as shown in Fig 1a The PGCL prepared in this study wasrubber-like elastic to show an extension over 250 and a full recovery as observed

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1152 I K Kwon et al

Table 1Characteristics of PGCL copolymer prepared

Mole ratio Composition a Yield Tgb Tc

b Tmb Mw

c acuteinhd

in feed of copolymer () ( plusmnC) ( plusmnC) ( plusmnC) (g dliexcl1)[G][CL] [G][CL]

5050 5149 962 iexcl154 mdash mdash 103 000 093

a Measured by 1H-NMRb Measured by DSC at a heating rate of 10plusmnC miniexcl1c Measured by GPC in chloroform at 1 ml miniexcl1 30plusmnCd Inherent viscosity in HFIP at 05 g dliexcl1 at 25plusmnC

(a)

(b)

Figure 1 Mechanical strength of PGCL (51 49) and PGLA (70 30) scaffolds (a) stressndashstraincurve of PGCL and PGLA and (b) elastic recovery of PGCL

in Fig 1b Porous PGCL matrices were prepared by a solvent-casting particulateleaching method using sodium chloride particles

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Fibroblast culture on surface-modied PGCL 1153

Preparation of PGCL scaffolds

The effects of the weight fraction and particle size of sodium chloride on theporosity pore diameter and surface volume ratio were investigated First of all thematrix porosity increased with increasing the weight fraction of sodium chlorideThe mean pore diameter increased too as the salt particle size increased The poresize was evaluated by a boundary area calculation method from a SEM picture [31]The mean pore sizes were 22 70 150 and 300 sup1m respectively for particle sizes of0 lt d lt 50 50 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m Membranesprepared with salt weight fractions above 90 wt had a uniform pore distribution asobserved by SEM however those with 70 or 80 wt formed asymmetric structureswith a dense impermeable skin at the bottom surface of the scaffolds

Degradation study of PGCL scaffolds

The degradation study of PGCL lm and scaffold (made with 95 wt salt poresize 150 sup1m) was conducted in PBS at 37plusmnC with a shaking incubator (100 rpm)The weight loss and molecular weight by GPC were measured as shown in Fig 2The weight of PGCL lm did not change up to 2 weeks but decreased graduallyand rapidly after 3 weeks At 6 weeks 60 of the initial mass was degraded Themolecular weights of PGCL lm and scaffold were rapidly reduced throughout thewhole period as shown in Fig 2b The smaller initial molecular weight of thescaffold than that of the lm may be caused by partial hydrolysis at the immersingprocess in water during scaffold preparation This result indicated that the PGCLcopolymer would be degraded mostly in 8ndash10 weeks to leave low Mw fractions

Surface hydrolysis and determination of the density of collagen coated

To enhance the hydrophilicity of the surface by introducing free carboxylic andhydroxyl end groups the PGCL scaffold was partially hydrolyzed by soaking inaqueous 1 N NaOH solution In addition the PGCL lm was treated under thesame conditions for ESCA and contact angle study Figure 3 shows the surfacemorphology of the PGCL scaffolds unmodi ed and hydrolyzed for 1 3 5 10 and20 min respectively It was revealed that the hydrolysis caused surface erosionresulting in increased pore size In addition the wettability of the PGCL wassigni cantly increased on hydrolysis The static contact angles of the unmodi edPGCL lm and the surface-hydrolyzed ones were measured by sessile drop methodas shown in Fig 4 The contact angle was gradually reduced with increasingtime to indicate approximately 65 deg after 3 min which was a substantiallydecreased value from 84 deg for the untreated one The changes of the surfaceelemental compositions of the hydrolyzed lms were analyzed by ESCA Thesurface composition of unmodi ed PGCL lm indicated 76 carbon and 24oxygen After hydrolysis they changed to 73 carbon and 27 oxygen as shownin Table 2 The increase in oxygen content on the surface-hydrolyzed PGCL lmshould be a result of the carboxylic and hydroxy groups evolved by the hydrolysis

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1154 I K Kwon et al

(a)

(b)

Figure 2 Degradation behavior of PGCL (a) weight loss of lm and (b) molecular weight loss ofscaffold (-pound-) and lm (-F-)

Table 2Atomic compositionof PGCL by ESCA analysisafter hydrolyzation for 3 min

Samples C O OC

Untreated PGCL 7628 2372 031Hydrolyzed PGCL 7280 2720 037

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Fibroblast culture on surface-modied PGCL 1155

Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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1156 I K Kwon et al

(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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1158 I K Kwon et al

Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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Fibroblast culture on surface-modied PGCL 1149

The polymerization reaction was carried at 170plusmnC in a silicone oil bath for 20 hThe copolymer was recovered by dissolving in CHCl3 and puri ed by precipitationin methanol (Fisher Co) For comparison a copolymer of glycolide and L-lactide(70 30 molmol) was copolymerized by the analogous method

Characterizations

The prepared PGCL copolymer was analyzed by 600 MHz 1H-NMR (VarianFT-NMR spectrometer) Hexa uoroacetone deuterate was used as solvent andtetramethyl silane (TMS) as an internal standard NMR spectra were obtained at7plusmnC PGCL (0072 g) was dissolved in 144 ml hexa uoroisopropanol (HFIP) andmeasured for inherent viscosity by an Ubbelohde viscometer The molecular weightof the PGCL copolymer was determined by gel permeable chromatography (GPCModel 510 pump equipped with a 410 Differential Refractometer) using threemicro-styragel columns (CHR2 HR4 and HR5E) from 500 to 4 pound 106Aring connectedin series Tetrahydrofuran (THF) was eluted at a rate of 10 ml miniexcl1 at 40plusmnCCalibration was carried out using polystyrene standards (Shodex) over a molecularweight range of 130 pound 103 ndash 196 pound 106

Thermal analysis of the polymer was carried out on a differential scanningcalorimeter (DSC Du Pont TA 2000) employing a heating rate of 10plusmnC miniexcl1Tensile property was measured by Instron (Model 5567) Samples were cut into1 pound 1 cm for compression and tensile testing The specimens attached to cardboardusing epoxy glue were drawn at a constant rate (1 mm miniexcl1 )

Static water contact angles on PGCL surfaces were measured by the sessiledrop method using deionized distilled water by a goniometer (model G-1 ErmaInc) Chemical structural changes on the hydrolyzed surface of the sampleswere examined by electron spectra for chemical analysis (ESCA Surface ScienceInstrument model 2803-S)

Preparation of PGCL scaffolds

PGCL porous membranes were prepared by a solvent-casting particulate leachingtechnique [25] that consisted of the following steps

(1) The sieved NaCl particles were added into 5 wtwt PGCL solutions inchloroform and the vortexed dispersions were cast in glass Petri dishes (diameter5 cm) Five different polymer salt compositions were applied in this study 30702080 1090 595 and 397 by wt ratios while the salt size was xed at150 lt d lt 300 sup1m For the 595 wt ratio four different salt sizes were used0 lt d lt 53 53 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m (2) Thesolvent was allowed to evaporate from the covered Petri dish at room temperatureover 48 h and then completely by vacuum drying at room temperature for 24 h(3) The PGCLsalt composite membranes were immersed in distilled deionizedwater with stirring for 48 h (the water was changed at every 12 h) to leach out the

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1150 I K Kwon et al

salt (4) The salt-free PGCL membranes were air-dried for 24 h and then vacuum-dried for 48 h The scaffolds were stored in a desiccator under vacuum

Degradation study of PGCL scaffolds

The degradation study of PGCL scaffolds was conducted in phosphate-bufferedsaline (PBS) at pH 74 at 37plusmnC using a shaking incubator (100 rpm) The pH of themedium was continuously monitored and the medium was Freshly replaced every48 h Weight loss and molecular weights were measured by GPC at certain timeperiods

Surface modi cation

For the hydrolysis the lms and scaffolds were placed in 1 N NaOH solution withshaking for 1 3 5 10 and 20 min respectively They were rinsed with waterextensively until the rinsing water became neutral nally washed with ethanol andfreeze-dried The elemental compositions of carbon and oxygen on the surfaceof the PGCL lms were obtained by ESCA Collagen (Type I Sigma C3929)was dissolved in 0001 N HCl solution to 01 wt concentration The scaffoldswere immersed in 70 ethanol for 5 min and then into the collagen solutions for30 min [28] and dried under vacuum for 24 h In order to decrease the degradationrate of the collagen by cross-linking the coated scaffolds were irradiated by UV254 nm at 4plusmnC for 2 h The specimens were exposed to eight surrounding 10-W UVlamps (Daeil DBO231S) and the distance between the light source and sample was5 in [28 37] Finally they were washed with PBS until the rinsing water becameneutral

Determination of the amount of type I collagen coated

The amount of collagen coated was assayed by the Bradford method [29] Inaddition it was con rmed whether the collagen coated was practically cross-linkedby UV irradiation The polymer sponges were incubated in Dulbeccorsquos modi edEaglersquos medium (DMEM Sigma) without fetal bovine serum (FBS) for 0 and 24 hin a CO2 incubator After incubation the polymer sponge was placed in 6 N HClsolution at 120plusmnC while the coated collagen was separated from the sponge anddissolved into the solution The absorbance of the solution was measured at 610 nmby an enzyme-linked immunosorbent assay (ELISA) apparatus (Spectra Max 340Molecular Device Inc CA USA)

Cell culture studies

Fibroblasts (NIH3T3 ATCC)were harvested after trypsin treatment (005 trypsin 002 EDTA Gibco) and cultured in DMEM supplemented with 10 bovine calfserum and 50 U mliexcl1 of penicillin and streptomycin (Sigma) The cells were main-tained at 37plusmnC in humidi ed 5 CO2 atmosphere All the data were presented as

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Fibroblast culture on surface-modied PGCL 1151

the mean values of three counts All the scaffolds were sterilized by UV irradia-tion for 5 min and immersed in 70 ethanol overnight and then washed with PBSthree times Cell suspensions (50 sup1l) containing a certain number of cells (5 pound 105

cells mliexcl1 for the porosity study 1 pound 106 cells mliexcl1 for the pore size study and5 pound 106 cells mliexcl1 for the surface modi cation study) were seeded onto polymerscaffolds (5 pound 5 pound 12 mm) located in 24-well tissue culture plates After 3 h 1 mlof culture medium was added to each well Cell adhesion and proliferation on thescaffolds were determined at 1 and 3 days after seeding The matrices were rinsedwith PBS to remove unattached cells before harvesting the adherent cells and cellcounting Cell numbers were determined by a uorometric quanti cation of DNAin the scaffolds by an assay according to West et al [30] An aliquot was read u-orometrically (Hoefer Scienti c Instruments DNA Fluorometer) using Hoechst dyeno 33258 (Polyscience Inc) Fluorescence emissions at 455 nm of the supernatantsamples were read with the excitation set at 350 nm The amount of DNA wasextrapolated from the standard curve of broblast (NIH3T3) DNA

Fibroblasts seeded onto PGCL scaffolds were xed for examination by scanningelectron microscope (SEM Stereoscan 360 Cambridge Instruments UK) Thescaffolds were washed with PBS and then xed in PBS containing 2 glutaldehydeat room temperature for 1 h followed by a incubation for 24 h at 4plusmnC After washingagain with PBS buffer the scaffolds were dehydrated through a graded series of 5060 70 80 90 and 100 ethanol each time for 10 min The samples were dried andcoated with an ultra-thin gold layer (100 Aring)

Statistical analysis

All the quantitative results were obtained from triplicate samples Data wereexpressed as a mean sect SD Statistical analysis was carried out using the unpairedStudentrsquos t-test A value of p lt 005 was considered to be statistically signi cant

RESULTS

Characterization of poly(glycolide-co--caprolactone) (PGCL)

The copolymer was characterized in terms of the thermal properties and microstruc-ture by means of viscosimetry DSC NMR spectroscopy and GPC Table 1 showsthat the PGCL had a molar composition (G CL) of 51 49 by NMR The mole-cular weight (Mw ) was measured at 103 000 by GPC and the intrinsic viscositywas 093 dl giexcl1 in chloroform The glass transition temperature (Tg ) was observedat iexcl154plusmnC but no crystallization temperature (Tc) or melting point (Tm ) to indi-cate the PGCL synthesized to be random and amorphous The PGCL revealed amuch lower tensile modulus but higher elongation than PGLA (70 30) (Mw wasabout 300 000) did as shown in Fig 1a The PGCL prepared in this study wasrubber-like elastic to show an extension over 250 and a full recovery as observed

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1152 I K Kwon et al

Table 1Characteristics of PGCL copolymer prepared

Mole ratio Composition a Yield Tgb Tc

b Tmb Mw

c acuteinhd

in feed of copolymer () ( plusmnC) ( plusmnC) ( plusmnC) (g dliexcl1)[G][CL] [G][CL]

5050 5149 962 iexcl154 mdash mdash 103 000 093

a Measured by 1H-NMRb Measured by DSC at a heating rate of 10plusmnC miniexcl1c Measured by GPC in chloroform at 1 ml miniexcl1 30plusmnCd Inherent viscosity in HFIP at 05 g dliexcl1 at 25plusmnC

(a)

(b)

Figure 1 Mechanical strength of PGCL (51 49) and PGLA (70 30) scaffolds (a) stressndashstraincurve of PGCL and PGLA and (b) elastic recovery of PGCL

in Fig 1b Porous PGCL matrices were prepared by a solvent-casting particulateleaching method using sodium chloride particles

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Fibroblast culture on surface-modied PGCL 1153

Preparation of PGCL scaffolds

The effects of the weight fraction and particle size of sodium chloride on theporosity pore diameter and surface volume ratio were investigated First of all thematrix porosity increased with increasing the weight fraction of sodium chlorideThe mean pore diameter increased too as the salt particle size increased The poresize was evaluated by a boundary area calculation method from a SEM picture [31]The mean pore sizes were 22 70 150 and 300 sup1m respectively for particle sizes of0 lt d lt 50 50 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m Membranesprepared with salt weight fractions above 90 wt had a uniform pore distribution asobserved by SEM however those with 70 or 80 wt formed asymmetric structureswith a dense impermeable skin at the bottom surface of the scaffolds

Degradation study of PGCL scaffolds

The degradation study of PGCL lm and scaffold (made with 95 wt salt poresize 150 sup1m) was conducted in PBS at 37plusmnC with a shaking incubator (100 rpm)The weight loss and molecular weight by GPC were measured as shown in Fig 2The weight of PGCL lm did not change up to 2 weeks but decreased graduallyand rapidly after 3 weeks At 6 weeks 60 of the initial mass was degraded Themolecular weights of PGCL lm and scaffold were rapidly reduced throughout thewhole period as shown in Fig 2b The smaller initial molecular weight of thescaffold than that of the lm may be caused by partial hydrolysis at the immersingprocess in water during scaffold preparation This result indicated that the PGCLcopolymer would be degraded mostly in 8ndash10 weeks to leave low Mw fractions

Surface hydrolysis and determination of the density of collagen coated

To enhance the hydrophilicity of the surface by introducing free carboxylic andhydroxyl end groups the PGCL scaffold was partially hydrolyzed by soaking inaqueous 1 N NaOH solution In addition the PGCL lm was treated under thesame conditions for ESCA and contact angle study Figure 3 shows the surfacemorphology of the PGCL scaffolds unmodi ed and hydrolyzed for 1 3 5 10 and20 min respectively It was revealed that the hydrolysis caused surface erosionresulting in increased pore size In addition the wettability of the PGCL wassigni cantly increased on hydrolysis The static contact angles of the unmodi edPGCL lm and the surface-hydrolyzed ones were measured by sessile drop methodas shown in Fig 4 The contact angle was gradually reduced with increasingtime to indicate approximately 65 deg after 3 min which was a substantiallydecreased value from 84 deg for the untreated one The changes of the surfaceelemental compositions of the hydrolyzed lms were analyzed by ESCA Thesurface composition of unmodi ed PGCL lm indicated 76 carbon and 24oxygen After hydrolysis they changed to 73 carbon and 27 oxygen as shownin Table 2 The increase in oxygen content on the surface-hydrolyzed PGCL lmshould be a result of the carboxylic and hydroxy groups evolved by the hydrolysis

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1154 I K Kwon et al

(a)

(b)

Figure 2 Degradation behavior of PGCL (a) weight loss of lm and (b) molecular weight loss ofscaffold (-pound-) and lm (-F-)

Table 2Atomic compositionof PGCL by ESCA analysisafter hydrolyzation for 3 min

Samples C O OC

Untreated PGCL 7628 2372 031Hydrolyzed PGCL 7280 2720 037

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Fibroblast culture on surface-modied PGCL 1155

Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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1156 I K Kwon et al

(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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1158 I K Kwon et al

Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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1150 I K Kwon et al

salt (4) The salt-free PGCL membranes were air-dried for 24 h and then vacuum-dried for 48 h The scaffolds were stored in a desiccator under vacuum

Degradation study of PGCL scaffolds

The degradation study of PGCL scaffolds was conducted in phosphate-bufferedsaline (PBS) at pH 74 at 37plusmnC using a shaking incubator (100 rpm) The pH of themedium was continuously monitored and the medium was Freshly replaced every48 h Weight loss and molecular weights were measured by GPC at certain timeperiods

Surface modi cation

For the hydrolysis the lms and scaffolds were placed in 1 N NaOH solution withshaking for 1 3 5 10 and 20 min respectively They were rinsed with waterextensively until the rinsing water became neutral nally washed with ethanol andfreeze-dried The elemental compositions of carbon and oxygen on the surfaceof the PGCL lms were obtained by ESCA Collagen (Type I Sigma C3929)was dissolved in 0001 N HCl solution to 01 wt concentration The scaffoldswere immersed in 70 ethanol for 5 min and then into the collagen solutions for30 min [28] and dried under vacuum for 24 h In order to decrease the degradationrate of the collagen by cross-linking the coated scaffolds were irradiated by UV254 nm at 4plusmnC for 2 h The specimens were exposed to eight surrounding 10-W UVlamps (Daeil DBO231S) and the distance between the light source and sample was5 in [28 37] Finally they were washed with PBS until the rinsing water becameneutral

Determination of the amount of type I collagen coated

The amount of collagen coated was assayed by the Bradford method [29] Inaddition it was con rmed whether the collagen coated was practically cross-linkedby UV irradiation The polymer sponges were incubated in Dulbeccorsquos modi edEaglersquos medium (DMEM Sigma) without fetal bovine serum (FBS) for 0 and 24 hin a CO2 incubator After incubation the polymer sponge was placed in 6 N HClsolution at 120plusmnC while the coated collagen was separated from the sponge anddissolved into the solution The absorbance of the solution was measured at 610 nmby an enzyme-linked immunosorbent assay (ELISA) apparatus (Spectra Max 340Molecular Device Inc CA USA)

Cell culture studies

Fibroblasts (NIH3T3 ATCC)were harvested after trypsin treatment (005 trypsin 002 EDTA Gibco) and cultured in DMEM supplemented with 10 bovine calfserum and 50 U mliexcl1 of penicillin and streptomycin (Sigma) The cells were main-tained at 37plusmnC in humidi ed 5 CO2 atmosphere All the data were presented as

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Fibroblast culture on surface-modied PGCL 1151

the mean values of three counts All the scaffolds were sterilized by UV irradia-tion for 5 min and immersed in 70 ethanol overnight and then washed with PBSthree times Cell suspensions (50 sup1l) containing a certain number of cells (5 pound 105

cells mliexcl1 for the porosity study 1 pound 106 cells mliexcl1 for the pore size study and5 pound 106 cells mliexcl1 for the surface modi cation study) were seeded onto polymerscaffolds (5 pound 5 pound 12 mm) located in 24-well tissue culture plates After 3 h 1 mlof culture medium was added to each well Cell adhesion and proliferation on thescaffolds were determined at 1 and 3 days after seeding The matrices were rinsedwith PBS to remove unattached cells before harvesting the adherent cells and cellcounting Cell numbers were determined by a uorometric quanti cation of DNAin the scaffolds by an assay according to West et al [30] An aliquot was read u-orometrically (Hoefer Scienti c Instruments DNA Fluorometer) using Hoechst dyeno 33258 (Polyscience Inc) Fluorescence emissions at 455 nm of the supernatantsamples were read with the excitation set at 350 nm The amount of DNA wasextrapolated from the standard curve of broblast (NIH3T3) DNA

Fibroblasts seeded onto PGCL scaffolds were xed for examination by scanningelectron microscope (SEM Stereoscan 360 Cambridge Instruments UK) Thescaffolds were washed with PBS and then xed in PBS containing 2 glutaldehydeat room temperature for 1 h followed by a incubation for 24 h at 4plusmnC After washingagain with PBS buffer the scaffolds were dehydrated through a graded series of 5060 70 80 90 and 100 ethanol each time for 10 min The samples were dried andcoated with an ultra-thin gold layer (100 Aring)

Statistical analysis

All the quantitative results were obtained from triplicate samples Data wereexpressed as a mean sect SD Statistical analysis was carried out using the unpairedStudentrsquos t-test A value of p lt 005 was considered to be statistically signi cant

RESULTS

Characterization of poly(glycolide-co--caprolactone) (PGCL)

The copolymer was characterized in terms of the thermal properties and microstruc-ture by means of viscosimetry DSC NMR spectroscopy and GPC Table 1 showsthat the PGCL had a molar composition (G CL) of 51 49 by NMR The mole-cular weight (Mw ) was measured at 103 000 by GPC and the intrinsic viscositywas 093 dl giexcl1 in chloroform The glass transition temperature (Tg ) was observedat iexcl154plusmnC but no crystallization temperature (Tc) or melting point (Tm ) to indi-cate the PGCL synthesized to be random and amorphous The PGCL revealed amuch lower tensile modulus but higher elongation than PGLA (70 30) (Mw wasabout 300 000) did as shown in Fig 1a The PGCL prepared in this study wasrubber-like elastic to show an extension over 250 and a full recovery as observed

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1152 I K Kwon et al

Table 1Characteristics of PGCL copolymer prepared

Mole ratio Composition a Yield Tgb Tc

b Tmb Mw

c acuteinhd

in feed of copolymer () ( plusmnC) ( plusmnC) ( plusmnC) (g dliexcl1)[G][CL] [G][CL]

5050 5149 962 iexcl154 mdash mdash 103 000 093

a Measured by 1H-NMRb Measured by DSC at a heating rate of 10plusmnC miniexcl1c Measured by GPC in chloroform at 1 ml miniexcl1 30plusmnCd Inherent viscosity in HFIP at 05 g dliexcl1 at 25plusmnC

(a)

(b)

Figure 1 Mechanical strength of PGCL (51 49) and PGLA (70 30) scaffolds (a) stressndashstraincurve of PGCL and PGLA and (b) elastic recovery of PGCL

in Fig 1b Porous PGCL matrices were prepared by a solvent-casting particulateleaching method using sodium chloride particles

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Fibroblast culture on surface-modied PGCL 1153

Preparation of PGCL scaffolds

The effects of the weight fraction and particle size of sodium chloride on theporosity pore diameter and surface volume ratio were investigated First of all thematrix porosity increased with increasing the weight fraction of sodium chlorideThe mean pore diameter increased too as the salt particle size increased The poresize was evaluated by a boundary area calculation method from a SEM picture [31]The mean pore sizes were 22 70 150 and 300 sup1m respectively for particle sizes of0 lt d lt 50 50 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m Membranesprepared with salt weight fractions above 90 wt had a uniform pore distribution asobserved by SEM however those with 70 or 80 wt formed asymmetric structureswith a dense impermeable skin at the bottom surface of the scaffolds

Degradation study of PGCL scaffolds

The degradation study of PGCL lm and scaffold (made with 95 wt salt poresize 150 sup1m) was conducted in PBS at 37plusmnC with a shaking incubator (100 rpm)The weight loss and molecular weight by GPC were measured as shown in Fig 2The weight of PGCL lm did not change up to 2 weeks but decreased graduallyand rapidly after 3 weeks At 6 weeks 60 of the initial mass was degraded Themolecular weights of PGCL lm and scaffold were rapidly reduced throughout thewhole period as shown in Fig 2b The smaller initial molecular weight of thescaffold than that of the lm may be caused by partial hydrolysis at the immersingprocess in water during scaffold preparation This result indicated that the PGCLcopolymer would be degraded mostly in 8ndash10 weeks to leave low Mw fractions

Surface hydrolysis and determination of the density of collagen coated

To enhance the hydrophilicity of the surface by introducing free carboxylic andhydroxyl end groups the PGCL scaffold was partially hydrolyzed by soaking inaqueous 1 N NaOH solution In addition the PGCL lm was treated under thesame conditions for ESCA and contact angle study Figure 3 shows the surfacemorphology of the PGCL scaffolds unmodi ed and hydrolyzed for 1 3 5 10 and20 min respectively It was revealed that the hydrolysis caused surface erosionresulting in increased pore size In addition the wettability of the PGCL wassigni cantly increased on hydrolysis The static contact angles of the unmodi edPGCL lm and the surface-hydrolyzed ones were measured by sessile drop methodas shown in Fig 4 The contact angle was gradually reduced with increasingtime to indicate approximately 65 deg after 3 min which was a substantiallydecreased value from 84 deg for the untreated one The changes of the surfaceelemental compositions of the hydrolyzed lms were analyzed by ESCA Thesurface composition of unmodi ed PGCL lm indicated 76 carbon and 24oxygen After hydrolysis they changed to 73 carbon and 27 oxygen as shownin Table 2 The increase in oxygen content on the surface-hydrolyzed PGCL lmshould be a result of the carboxylic and hydroxy groups evolved by the hydrolysis

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1154 I K Kwon et al

(a)

(b)

Figure 2 Degradation behavior of PGCL (a) weight loss of lm and (b) molecular weight loss ofscaffold (-pound-) and lm (-F-)

Table 2Atomic compositionof PGCL by ESCA analysisafter hydrolyzation for 3 min

Samples C O OC

Untreated PGCL 7628 2372 031Hydrolyzed PGCL 7280 2720 037

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Fibroblast culture on surface-modied PGCL 1155

Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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Fibroblast culture on surface-modied PGCL 1151

the mean values of three counts All the scaffolds were sterilized by UV irradia-tion for 5 min and immersed in 70 ethanol overnight and then washed with PBSthree times Cell suspensions (50 sup1l) containing a certain number of cells (5 pound 105

cells mliexcl1 for the porosity study 1 pound 106 cells mliexcl1 for the pore size study and5 pound 106 cells mliexcl1 for the surface modi cation study) were seeded onto polymerscaffolds (5 pound 5 pound 12 mm) located in 24-well tissue culture plates After 3 h 1 mlof culture medium was added to each well Cell adhesion and proliferation on thescaffolds were determined at 1 and 3 days after seeding The matrices were rinsedwith PBS to remove unattached cells before harvesting the adherent cells and cellcounting Cell numbers were determined by a uorometric quanti cation of DNAin the scaffolds by an assay according to West et al [30] An aliquot was read u-orometrically (Hoefer Scienti c Instruments DNA Fluorometer) using Hoechst dyeno 33258 (Polyscience Inc) Fluorescence emissions at 455 nm of the supernatantsamples were read with the excitation set at 350 nm The amount of DNA wasextrapolated from the standard curve of broblast (NIH3T3) DNA

Fibroblasts seeded onto PGCL scaffolds were xed for examination by scanningelectron microscope (SEM Stereoscan 360 Cambridge Instruments UK) Thescaffolds were washed with PBS and then xed in PBS containing 2 glutaldehydeat room temperature for 1 h followed by a incubation for 24 h at 4plusmnC After washingagain with PBS buffer the scaffolds were dehydrated through a graded series of 5060 70 80 90 and 100 ethanol each time for 10 min The samples were dried andcoated with an ultra-thin gold layer (100 Aring)

Statistical analysis

All the quantitative results were obtained from triplicate samples Data wereexpressed as a mean sect SD Statistical analysis was carried out using the unpairedStudentrsquos t-test A value of p lt 005 was considered to be statistically signi cant

RESULTS

Characterization of poly(glycolide-co--caprolactone) (PGCL)

The copolymer was characterized in terms of the thermal properties and microstruc-ture by means of viscosimetry DSC NMR spectroscopy and GPC Table 1 showsthat the PGCL had a molar composition (G CL) of 51 49 by NMR The mole-cular weight (Mw ) was measured at 103 000 by GPC and the intrinsic viscositywas 093 dl giexcl1 in chloroform The glass transition temperature (Tg ) was observedat iexcl154plusmnC but no crystallization temperature (Tc) or melting point (Tm ) to indi-cate the PGCL synthesized to be random and amorphous The PGCL revealed amuch lower tensile modulus but higher elongation than PGLA (70 30) (Mw wasabout 300 000) did as shown in Fig 1a The PGCL prepared in this study wasrubber-like elastic to show an extension over 250 and a full recovery as observed

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Table 1Characteristics of PGCL copolymer prepared

Mole ratio Composition a Yield Tgb Tc

b Tmb Mw

c acuteinhd

in feed of copolymer () ( plusmnC) ( plusmnC) ( plusmnC) (g dliexcl1)[G][CL] [G][CL]

5050 5149 962 iexcl154 mdash mdash 103 000 093

a Measured by 1H-NMRb Measured by DSC at a heating rate of 10plusmnC miniexcl1c Measured by GPC in chloroform at 1 ml miniexcl1 30plusmnCd Inherent viscosity in HFIP at 05 g dliexcl1 at 25plusmnC

(a)

(b)

Figure 1 Mechanical strength of PGCL (51 49) and PGLA (70 30) scaffolds (a) stressndashstraincurve of PGCL and PGLA and (b) elastic recovery of PGCL

in Fig 1b Porous PGCL matrices were prepared by a solvent-casting particulateleaching method using sodium chloride particles

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Preparation of PGCL scaffolds

The effects of the weight fraction and particle size of sodium chloride on theporosity pore diameter and surface volume ratio were investigated First of all thematrix porosity increased with increasing the weight fraction of sodium chlorideThe mean pore diameter increased too as the salt particle size increased The poresize was evaluated by a boundary area calculation method from a SEM picture [31]The mean pore sizes were 22 70 150 and 300 sup1m respectively for particle sizes of0 lt d lt 50 50 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m Membranesprepared with salt weight fractions above 90 wt had a uniform pore distribution asobserved by SEM however those with 70 or 80 wt formed asymmetric structureswith a dense impermeable skin at the bottom surface of the scaffolds

Degradation study of PGCL scaffolds

The degradation study of PGCL lm and scaffold (made with 95 wt salt poresize 150 sup1m) was conducted in PBS at 37plusmnC with a shaking incubator (100 rpm)The weight loss and molecular weight by GPC were measured as shown in Fig 2The weight of PGCL lm did not change up to 2 weeks but decreased graduallyand rapidly after 3 weeks At 6 weeks 60 of the initial mass was degraded Themolecular weights of PGCL lm and scaffold were rapidly reduced throughout thewhole period as shown in Fig 2b The smaller initial molecular weight of thescaffold than that of the lm may be caused by partial hydrolysis at the immersingprocess in water during scaffold preparation This result indicated that the PGCLcopolymer would be degraded mostly in 8ndash10 weeks to leave low Mw fractions

Surface hydrolysis and determination of the density of collagen coated

To enhance the hydrophilicity of the surface by introducing free carboxylic andhydroxyl end groups the PGCL scaffold was partially hydrolyzed by soaking inaqueous 1 N NaOH solution In addition the PGCL lm was treated under thesame conditions for ESCA and contact angle study Figure 3 shows the surfacemorphology of the PGCL scaffolds unmodi ed and hydrolyzed for 1 3 5 10 and20 min respectively It was revealed that the hydrolysis caused surface erosionresulting in increased pore size In addition the wettability of the PGCL wassigni cantly increased on hydrolysis The static contact angles of the unmodi edPGCL lm and the surface-hydrolyzed ones were measured by sessile drop methodas shown in Fig 4 The contact angle was gradually reduced with increasingtime to indicate approximately 65 deg after 3 min which was a substantiallydecreased value from 84 deg for the untreated one The changes of the surfaceelemental compositions of the hydrolyzed lms were analyzed by ESCA Thesurface composition of unmodi ed PGCL lm indicated 76 carbon and 24oxygen After hydrolysis they changed to 73 carbon and 27 oxygen as shownin Table 2 The increase in oxygen content on the surface-hydrolyzed PGCL lmshould be a result of the carboxylic and hydroxy groups evolved by the hydrolysis

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1154 I K Kwon et al

(a)

(b)

Figure 2 Degradation behavior of PGCL (a) weight loss of lm and (b) molecular weight loss ofscaffold (-pound-) and lm (-F-)

Table 2Atomic compositionof PGCL by ESCA analysisafter hydrolyzation for 3 min

Samples C O OC

Untreated PGCL 7628 2372 031Hydrolyzed PGCL 7280 2720 037

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Fibroblast culture on surface-modied PGCL 1155

Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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1156 I K Kwon et al

(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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Table 1Characteristics of PGCL copolymer prepared

Mole ratio Composition a Yield Tgb Tc

b Tmb Mw

c acuteinhd

in feed of copolymer () ( plusmnC) ( plusmnC) ( plusmnC) (g dliexcl1)[G][CL] [G][CL]

5050 5149 962 iexcl154 mdash mdash 103 000 093

a Measured by 1H-NMRb Measured by DSC at a heating rate of 10plusmnC miniexcl1c Measured by GPC in chloroform at 1 ml miniexcl1 30plusmnCd Inherent viscosity in HFIP at 05 g dliexcl1 at 25plusmnC

(a)

(b)

Figure 1 Mechanical strength of PGCL (51 49) and PGLA (70 30) scaffolds (a) stressndashstraincurve of PGCL and PGLA and (b) elastic recovery of PGCL

in Fig 1b Porous PGCL matrices were prepared by a solvent-casting particulateleaching method using sodium chloride particles

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Fibroblast culture on surface-modied PGCL 1153

Preparation of PGCL scaffolds

The effects of the weight fraction and particle size of sodium chloride on theporosity pore diameter and surface volume ratio were investigated First of all thematrix porosity increased with increasing the weight fraction of sodium chlorideThe mean pore diameter increased too as the salt particle size increased The poresize was evaluated by a boundary area calculation method from a SEM picture [31]The mean pore sizes were 22 70 150 and 300 sup1m respectively for particle sizes of0 lt d lt 50 50 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m Membranesprepared with salt weight fractions above 90 wt had a uniform pore distribution asobserved by SEM however those with 70 or 80 wt formed asymmetric structureswith a dense impermeable skin at the bottom surface of the scaffolds

Degradation study of PGCL scaffolds

The degradation study of PGCL lm and scaffold (made with 95 wt salt poresize 150 sup1m) was conducted in PBS at 37plusmnC with a shaking incubator (100 rpm)The weight loss and molecular weight by GPC were measured as shown in Fig 2The weight of PGCL lm did not change up to 2 weeks but decreased graduallyand rapidly after 3 weeks At 6 weeks 60 of the initial mass was degraded Themolecular weights of PGCL lm and scaffold were rapidly reduced throughout thewhole period as shown in Fig 2b The smaller initial molecular weight of thescaffold than that of the lm may be caused by partial hydrolysis at the immersingprocess in water during scaffold preparation This result indicated that the PGCLcopolymer would be degraded mostly in 8ndash10 weeks to leave low Mw fractions

Surface hydrolysis and determination of the density of collagen coated

To enhance the hydrophilicity of the surface by introducing free carboxylic andhydroxyl end groups the PGCL scaffold was partially hydrolyzed by soaking inaqueous 1 N NaOH solution In addition the PGCL lm was treated under thesame conditions for ESCA and contact angle study Figure 3 shows the surfacemorphology of the PGCL scaffolds unmodi ed and hydrolyzed for 1 3 5 10 and20 min respectively It was revealed that the hydrolysis caused surface erosionresulting in increased pore size In addition the wettability of the PGCL wassigni cantly increased on hydrolysis The static contact angles of the unmodi edPGCL lm and the surface-hydrolyzed ones were measured by sessile drop methodas shown in Fig 4 The contact angle was gradually reduced with increasingtime to indicate approximately 65 deg after 3 min which was a substantiallydecreased value from 84 deg for the untreated one The changes of the surfaceelemental compositions of the hydrolyzed lms were analyzed by ESCA Thesurface composition of unmodi ed PGCL lm indicated 76 carbon and 24oxygen After hydrolysis they changed to 73 carbon and 27 oxygen as shownin Table 2 The increase in oxygen content on the surface-hydrolyzed PGCL lmshould be a result of the carboxylic and hydroxy groups evolved by the hydrolysis

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(a)

(b)

Figure 2 Degradation behavior of PGCL (a) weight loss of lm and (b) molecular weight loss ofscaffold (-pound-) and lm (-F-)

Table 2Atomic compositionof PGCL by ESCA analysisafter hydrolyzation for 3 min

Samples C O OC

Untreated PGCL 7628 2372 031Hydrolyzed PGCL 7280 2720 037

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Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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Fibroblast culture on surface-modied PGCL 1153

Preparation of PGCL scaffolds

The effects of the weight fraction and particle size of sodium chloride on theporosity pore diameter and surface volume ratio were investigated First of all thematrix porosity increased with increasing the weight fraction of sodium chlorideThe mean pore diameter increased too as the salt particle size increased The poresize was evaluated by a boundary area calculation method from a SEM picture [31]The mean pore sizes were 22 70 150 and 300 sup1m respectively for particle sizes of0 lt d lt 50 50 lt d lt 150 150 lt d lt 300 and 300 lt d lt 500 sup1m Membranesprepared with salt weight fractions above 90 wt had a uniform pore distribution asobserved by SEM however those with 70 or 80 wt formed asymmetric structureswith a dense impermeable skin at the bottom surface of the scaffolds

Degradation study of PGCL scaffolds

The degradation study of PGCL lm and scaffold (made with 95 wt salt poresize 150 sup1m) was conducted in PBS at 37plusmnC with a shaking incubator (100 rpm)The weight loss and molecular weight by GPC were measured as shown in Fig 2The weight of PGCL lm did not change up to 2 weeks but decreased graduallyand rapidly after 3 weeks At 6 weeks 60 of the initial mass was degraded Themolecular weights of PGCL lm and scaffold were rapidly reduced throughout thewhole period as shown in Fig 2b The smaller initial molecular weight of thescaffold than that of the lm may be caused by partial hydrolysis at the immersingprocess in water during scaffold preparation This result indicated that the PGCLcopolymer would be degraded mostly in 8ndash10 weeks to leave low Mw fractions

Surface hydrolysis and determination of the density of collagen coated

To enhance the hydrophilicity of the surface by introducing free carboxylic andhydroxyl end groups the PGCL scaffold was partially hydrolyzed by soaking inaqueous 1 N NaOH solution In addition the PGCL lm was treated under thesame conditions for ESCA and contact angle study Figure 3 shows the surfacemorphology of the PGCL scaffolds unmodi ed and hydrolyzed for 1 3 5 10 and20 min respectively It was revealed that the hydrolysis caused surface erosionresulting in increased pore size In addition the wettability of the PGCL wassigni cantly increased on hydrolysis The static contact angles of the unmodi edPGCL lm and the surface-hydrolyzed ones were measured by sessile drop methodas shown in Fig 4 The contact angle was gradually reduced with increasingtime to indicate approximately 65 deg after 3 min which was a substantiallydecreased value from 84 deg for the untreated one The changes of the surfaceelemental compositions of the hydrolyzed lms were analyzed by ESCA Thesurface composition of unmodi ed PGCL lm indicated 76 carbon and 24oxygen After hydrolysis they changed to 73 carbon and 27 oxygen as shownin Table 2 The increase in oxygen content on the surface-hydrolyzed PGCL lmshould be a result of the carboxylic and hydroxy groups evolved by the hydrolysis

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(a)

(b)

Figure 2 Degradation behavior of PGCL (a) weight loss of lm and (b) molecular weight loss ofscaffold (-pound-) and lm (-F-)

Table 2Atomic compositionof PGCL by ESCA analysisafter hydrolyzation for 3 min

Samples C O OC

Untreated PGCL 7628 2372 031Hydrolyzed PGCL 7280 2720 037

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Fibroblast culture on surface-modied PGCL 1155

Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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1160 I K Kwon et al

REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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(a)

(b)

Figure 2 Degradation behavior of PGCL (a) weight loss of lm and (b) molecular weight loss ofscaffold (-pound-) and lm (-F-)

Table 2Atomic compositionof PGCL by ESCA analysisafter hydrolyzation for 3 min

Samples C O OC

Untreated PGCL 7628 2372 031Hydrolyzed PGCL 7280 2720 037

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Fibroblast culture on surface-modied PGCL 1155

Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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Fibroblast culture on surface-modied PGCL 1155

Figure 3 SEM pictures of the surface morphologies of PGCL scaffolds hydrolyzed for various times(a) 1 min (b) 3 min (c) 5 min (d) 10 min and (e) 20 min

Figure 4 Contact angles of PGCL lms hydrolyzed for various periods

The density of type I collagen coated on the polymer sponge was measuredby Bradford assay after incubation for 24 h The amount of collagen was364 sect 115 sup1g mgiexcl1 initially and 245 sect 127 sup1g mgiexcl1 after incubation As thecollagen coated on the scaffolds exhibited the large amount even after incubationin the culture medium the UV cross-linking was effective The collagen coated isexpected to play an important role in encouraging cell adhesion and growth

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(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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(a)

(b)

Figure 5 Fibroblast culture on PGCL scaffolds (a) with various pore sizes made from 95 wtsalt fractions cell density was 1 pound 106 cellsml and (b) with various salt fractions made from150ndash300 sup1m salt cell density was 5 pound 105 cellsml

Cell culture studies

Fibroblasts were seeded on each scaffold to study the optimal porosity and poresize for cell culture First of all small pore-sized scaffolds demonstrated lowcell adhesion and little proliferation while large pore-sized ones revealed high celladhesion and proliferation as shown in Fig 5a The number of cells adhered andproliferated were the largest in the case of a 300-sup1m pore size at 15 sect 065 pound 104

cells per matrix at 1 day and 40 sect 044 pound 104 cells per matrix ie 3 days As shownin Fig 5b the higher the porosity the better the cell adhesion and proliferation Thecell density grown for 3 days on the matrix prepared from 95 wt salt fraction was24 sect 044 pound 104 cells per matrix ie twice as many as on 70 wt matrix (12 sect034 pound 104 cells per matrix) This might be due to the scaffold with a large pore

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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Fibroblast culture on surface-modied PGCL 1157

Figure 6 Fibroblast culture on various modi ed PGCL scaffolds

Figure 7 Morphologies of broblasts adhered at 1 day after cell seeding on (a) unmodi ed PGCL(b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen-coated

and high porosity yielding large a surface area and interconnection and thereforeproviding easy transport of media into the matrix

To evaluate the effect of the surface modi cations of PGCL scaffolds broblastswere seeded in the presence of serum on the surface-modi ed scaffolds hydrolyzedandor collagen coated The number of broblast attached on the scaffold wasmeasured by analyzing DNA assay as shown in Fig 6 The broblasts were

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

Dow

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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Figure 8 Morphologies of broblasts proliferated for 3 days after cell seeding on (a) unmodi edPGCL (b) hydrolyzed (c) collagen coated and (d) hydrolyzed and collagen coated

cultured relatively well on the PGCL scaffold unmodi ed as well as the controlin the absence of the scaffold However on the hydrolyzed scaffold the numberof adhered and proliferated broblasts increased to a great extent compared withthe untreated one Hydrophilic carboxylic and hydroxyl end groups evolved byhydrolysis might have enhanced the attachment of cells to the scaffold surface asmediated by serum protein [27 32] as shown in Figs 6 and 7 Collagen coatingexhibited further contribution to enhance the cell density although the differencewas not so large The cell morphology on the PGCL scaffolds examined bySEM also showed a difference After 1 day culture the cells on the unmodi edPGCL scaffold appeared mostly round and aggregated On the contrary broblastsappeared both as individual cells and as cell aggregates on the surface-modi edscaffolds as shown in Fig 7 After 3 days culture the individual cells appeared tobe well spread to yield ECM proteins especially in the case of the hydrolyzed andcollagen-coated scaffold (Fig 8) Such an improved cell adhesion and proliferationon the modi ed scaffolds should be a result of the increased wetting (hydrophilicity)due to hydrolysis In addition collagen appeared to contribute to greatly enhancecell interactions

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

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1160 I K Kwon et al

REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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June

201

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Fibroblast culture on surface-modied PGCL 1159

DISCUSSION

Biodegradable polymer matrices are attractive synthetic ECMs for tissue engineer-ing and cell transplantation because they act as a temporary matrix and are laterresorbed as seeded cells develop new tissue Poly(glycolide) and its lactide copoly-mers have attracted much interest because they have very low immunogenicity andare biodegradable in the human body Poly(-caprolactone) is a more exible ma-terial which has a low melting temperature (58plusmnC) a low crystallinity dependingon the Mw and good solubility in most organic solvents The random copolymerof glycolide and -caprolactone (51 49) applied here showed an elastic character-istic It degraded rapidly and was presumably complete in 8ndash10 weeks in vitroThe porosity pore size and macroscopic dimension of scaffolds are the most im-portant factors associated with cell proliferation For tissue engineering it is verynecessary to obtain a maximal supply of nutrition by diffusion into tissue culturemedia in vitro or through newly-formed blood vessels in vivo It was shown in thisstudy that the large pore size above 150 sup1m were more effective for a broblastculture than the smaller pore size In addition the higher the porosity the larger celldensity

The initial cell adhesion is an important step in a wide variety of biologicalprocesses It is dependent on the biocompatibility of synthetic implant materialsto play a key-role in tissue and organ formation and in the generation of traction forthe migration of cells The ability to predict and control the interaction of cells withnonbiological materials underlies the rational design of biocompatible implants andtissue-engineered bio-hybrid organs Cell culture systems that utilize polymericsupports in a medium containing serum or protein are affected by proteins adsorbedat material interfaces [33] Recently the complex interrelationships among materialsurface properties adsorbed proteins and cellular responses have been extensivelystudied [23 33ndash36] In this study it was demonstrated that the surface-hydrolyzedandor collagen-coated PGCL scaffolds revealed an enhanced initial adhesionand proliferation of broblast compared to untreated PGCL Fibroblasts werewell attached and spread on the surface-hydrolyzed and collagen-coated PGCLscaffold The strategy of optimizing cellndashbiomaterial interactions to increaseinitial cell seeding density may be very useful for engineering tissues of high celldensity

This modi ed porous scaffold was exible and elastic and easy to process andtherefore promising for the regeneration of tissues especially for soft tissues Aculture study using human bladder cells is in progress

Acknowledgement

This study was supported by the Biotech 2000 Program (Grant No 2N169302N18030 and 2N19830) of the Ministry of Science and Technology Korea

Dow

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1160 I K Kwon et al

REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

Dow

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ded

by [

Uni

vers

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f V

icto

ria]

at 0

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04

June

201

4

1160 I K Kwon et al

REFERENCES

1 I V Yannas Angew Chem Int Ed Engl 29 20 (1990)2 T Natsume O Ike T Okada N Takimoto Y Shimizu and Y Ikada J Biomed Mater Res 27

867 (1993)3 D A Grande M I Pitman L Peterson K Menche and M Klein J Orthop Res 7 208 (1989)4 J D Bronzino The Biomedical Engineering Handbook CRC Press Boca Raton FL (1995)5 D J Mooney C Breuer K McNamara J P Vacanti and R Langer Tissue Eng 1 107 (1995)6 D J Mooney C L Mazzoni C Breuer K McNamara D Hern J P Vacanti and R Langer

Biomaterials 17 115 (1996)7 A G Mikos G SarakinosS M Leite J P Vacanti and R Langer Biomaterials 14 323 (1993)8 J E Pons R A Clandinning and S Cohen Soc Plastr Eng Tech Pap 21 567 (1975)9 S C Woodward P S Brewer F Montarned A Schindler and C G Pitt J Biomed Mater Res

19 437 (1985)10 T Ziegler and R M Nerem J Cell Biochem 56 204 (1994)11 J Hirai and T Matsuda Cell Transplant 4 597 (1995)12 K H Stenzel T Miyata and A L Rubin Annu Rev Biophys Bioeng 3 231 (1974)13 A Atala W Kim K T Paige C A Vacanti and A Retik J Urol 152 641 (1994)14 L Shapiro and S Cohen Biomaterials 18 583 (1997)15 I Benedetti R Cortivo T Berti and F Rea Biomaterials 14 1154 (1993)16 V Dizit Artif Organs 18 371 (1994)17 R S Bezwada and A G Scopelianos U S Patent 5468253 (November 1995)18 D G Pitt M M Gratzl G L Kimmel J Surles and A Schindler Biomaterials 17 215 (1996)19 P van der Valk A W J van Pelt H J Busscher H P de Jong Ch R H Wildevuur and

J Arends J Biomed Mater Res 17 807 (1983)20 D W Grijpma G J Zondervan and A J Pennings Polym Bull 25 327 (1991)21 T Nakamura Y Shimizu Y Takimo T Tsuda Y Li T Kiyotani M Teramachi S Hyon

Y Ikada and K Nishiya J Biomed Mater Res 42 475 (1998)22 R P Lanza R Langer and W L Chick Principle of Tissue Engineering Academic Press

Landes Bioscience (1996)23 J A Hubbel BioTechnol 13 565 (1995)24 K Smetana Jr Biomaterials 14 1046 (1993)25 A G Mikos A J Thorsen L A Czerwonka Y Bao and R Langer Polymer 35 1068 (1994)26 J Gao L Niklason and R Langer J Biomed Mater Res 42 417 (1998)27 A Curtis and J Forrester J Cell Sci 71 17 (1984)28 M G Dunn P N Avasarala and J P Zawadsky J Biomed Mater Res 27 1545 (1993)29 M Bradford Anal Biochem 72 248 (1976)30 D C West A Sattar and S Kumar Anal Biochem 147 289 (1985)31 H van V Lawrence in Elements of Materials Science and Engineering 6th edn p 219

Addison-Wesley (1989)32 H M Kowalczynska and J Kaminski J Cell Biol 99 587 (1991)33 P B van Wachem T Beugeling J Feijen A Bantjes J P Detmers and W G van Aken

Biomaterials 6 403 (1985)34 H L Wald G Sarakinos M D Lyman A G Mikos J P Vacanti and R Langer Biomaterials

14 270 (1993)35 P B van Wachem A H Hogt T Beugeling J Feijen A Bantjes J P Detmers and W G van

Aken J Biol Chem 267 10133 (1992)36 K Webb B Hlady and P A Tresco J Biomed Mater Res 41 422 (1998)37 Y P Kato R A Christiansen Hahn S J Shieh J D Goldstein S A Lund and F H Silver

Biomaterials 10 38 (1989)

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