Degradation and cell culture studies on block copolymersprepared by ring opening polymerization of �-caprolactonein the presence of poly(ethylene glycol)

Ming-Hsi Huang,1 Suming Li,1 Dietmar Werner Hutmacher,2,3 Jan-Thorsten Schantz,2,4,5

Charles Alfred Vacanti,5,6 Christian Braud,1 Michel Vert1

1Centre de Recherche sur les Biopolymeres Artificiels, Faculte de Pharmacie, 15 avenue Charles Flahault,34060 Montpellier, France2Division of Bioengineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore3Department of Orthopedic Surgery, National University of Singapore, 10 Kent Ridge Crescent,Singapore 119260, Singapore4Department of Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore5Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School,Worcester, Massachusetts6Brigham and Women’s Hospital, Department of Anesthesiology, Perioperative and Pain Medicine, 75 Francis Street,CWN L1, Boston, Massachusetts 02115

Received 18 September 2003; revised 3 December 2003; accepted 16 January 2004Published online 23 March 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30008

Abstract: Poly(�-caprolactone) (PCL) and its block copoly-mers with poly(ethylene glycol) (PEG) were prepared byring-opening polymerization of �-caprolactone in the pres-ence of ethylene glycol or PEG, using zinc metal as catalyst.The resulting polymers were characterized by various ana-lytical techniques such as 1H NMR, SEC, DSC, IR, X-ray,ESEM, and CZE. PCL/PEG copolymers with long PCLchains presented the same crystalline structure as PCL ho-mopolymer, whereas PEG-bearing short PCL blocks re-tained the crystalline structure of PEG and exhibited anamphiphilic behavior in aqueous solutions. Degradation ofPCL and PCL/PEG diblock and triblock copolymers wasrealized in a 0.13 M, pH 7.4 phosphate buffer at 37°C. Theresults indicated that the copolymers exhibited higher hy-drophilicity and degradability compared with the PCL ho-mopolymer. Large amounts of PEG were released from the

bulk after 60 weeks’ degradation. In vitro cell culture studieswere conducted on scaffolds manufactured via solid freeform fabrication by using primary human and rat bonemarrow derived stromal cells (hMSC, rMSC). Light, scan-ning electron, and confocal laser microscopy, as well asimmunocytochemistry, showed cell attachment, prolifera-tion, and extracellular matrix production on the surface, aswell as inside the scaffold architecture. Copolymers showedbetter performance in the cell culture studies than the PCLhomopolymer. © 2004 Wiley Periodicals, Inc. J Biomed Ma-ter Res 69A: 417–427, 2004

Key words: poly(�-caprolactone); poly(ethylene glycol); hy-drolytic degradation; tissue engineering; bone marrow stro-mal cells

INTRODUCTION

The first generation of bioresorbable scaffolds fortissue-engineering applications have been fabricatedfrom synthetic polymers of the aliphatic polyesterfamily.1,2 Polymers such as poly(lactic acid) (PLA),poly(glycolic acid) (PGA), and poly(�-caprolactone)(PCL), were preferred because they had been used as

medical and drug delivery devices with Food andDrug Administration approval.3,4 However, the num-ber of bioresorbable polymers is very limited. Thus, alarger selection of polymers with variable properties isneeded for the design and fabrication of devices andscaffolds for various applications.5,6 In this regard,polyester-polyether block copolymers composed ofPCL or PLA and poly(ethylene glycol) (PEG) attractedmuch attention as they offered the possibility of vary-ing the ratio of hydrophobic/hydrophilic constituentsto modulate the degradability and hydrophilicity ofthe polymer matrix and surface.7–14 PCL/PEGdiblock, triblock, and star-shaped block copolymers

Correspondence to: Suming Li; e-mail: lisuming@univ-montp1.fr and Dietmar Werner Hutmacher; e-mail:biedwh@nus.edu.sg

© 2004 Wiley Periodicals, Inc.

were prepared by ring-opening polymerization of�-caprolactone in the presence of monohydroxyl, di-hydroxyl, or multiarm PEG.11,12 PCL/PEG multiblockcopolymers were synthesized by polycondensation ofPEG bearing two carboxyl endgroups and PCL diolsin the presence of dicyclohexylcarbodiimide (DCC) ascoupling agent.13,14 It was observed that incorporationof PEG improved the hydrophilicity of the multiblockcopolymers with respect to PCL homopolymer.14

However, in diblock and triblock copolymers, no de-tailed studies have been performed on the hydrolyticdegradation behaviors, which are of major importancefor biomedical applications.

In this work, PCL homopolymer and PCL/PEGdiblock and triblock copolymers were prepared byring-opening polymerization of �-caprolactone in thepresence of ethylene glycol or PEG. The resulted poly-mers were characterized and allowed to degrade in a0.13 M pH � 7.4 phosphate buffer. PEG-bearingoligo(�-caprolactone) chains, OCL-PEG and OCL-PEG-OCL, were synthesized to mimic the degradationproducts of PCL/PEG copolymers. On the other hand,scaffolds were fabricated via solid free form fabrica-tion (SFF), and preliminary cell culture studies wereperformed to evaluate the cell compatibility of thecopolymers in comparison with PCL.

MATERIALS AND METHODS

Polymerization

�-Caprolactone was purchased from Acros Organics(Noisy-Le-Grand, France). Ethylene glycol, monohydroxylPEG, and dihydroxyl PEG were supplied by Fluka (St.Quentin Fallavier, France). Zinc powder was obtained fromMerck (Darmstadt, Germany). These compounds were usedwithout further purification.

PCL homopolymer and PCL/PEG block copolymers weresynthesized by bulk ring-opening polymerization of �-cap-rolactone as previously reported.9 For the synthesis of PCLhomopolymer, �-caprolactone, zinc powder (0.05 wt %) andethylene glycol (EG) were introduced into a round-bot-tomed flask. Polymerization was realized under vacuum at140°C for 20 days. In PCL/PEG and OCL/PEG block copol-ymers, monohydroxyl PEG or dihydroxyl PEG was used asinitiator instead of EG. The reaction time was 7 days. All thepolymers were recovered by the dissolution/precipitationmethod with dichloromethane as solvent and ethanol asnonsolvent, followed by filtration and vacuum drying up toconstant weight.

Hydrolytic degradation

PCL, PCL-PEG, and PCL-PEG-PCL were processed in theform of circular films of 75-mm diameter and 0.4-mm thick-

ness by compression molding at 100°C and 150 bars. Spec-imens measuring 10 mm � 10 mm � 0.4 mm were then cutfrom the films.

The specimens were introduced into small flasks filledwith 5 mL of 0.13 M, pH 7.4, phosphate buffer containing0.02% NaN3. Degradation was performed at 37°C. At eachdegradation time point, three specimens were withdrawnand washed with distilled water. After wiping, films wereweighed and then vacuum dried to constant weight beforebeing subjected to various analyses.

Measurements

1H nuclear magnetic resonance (1H NMR) spectra wererecorded at room temperature with a Bruker spectrometeroperating at 250 MHz using deuterated chloroform as sol-vent. Size-exclusion chromatography (SEC) was performedon a Waters 410 differential refractometer and a PLgel 5-�mmixed-C 60-cm column, the mobile phase being tetrahydro-furan (THF) and the flow rate 1 mL/min. Calibration wasaccomplished with Polysciences polystyrene standards. In-frared spectroscopy (IR) spectra were recorded from a Per-kin Elmer 1760 FTIR spectrometer. Films were cast on aNaCl plate from THF solutions. Differential scanning calo-rimetry (DSC) measurements were conducted under nitro-gen atmosphere on a Perkin Elmer DSC6 calorimeter. Thesamples were heated from 20°C to 105°C at 5°C/min. X-raydiffraction spectra were obtained with a Philips apparatususing a Cu K� source (0.154 nm). Capillary zone electro-phoresis (CZE) data were collected by using a P/ACE 5000Beckman instrument equipped with UV absorbance detec-tion at 254 nm and a fused-silica capillary (ID 50 �m, length57 cm) with reverse mode.19

Scaffold fabrication

Scaffolds were fabricated by using a rapid prototyping(RP) machine built in house at the Freiburg Materials Re-search Center (Albert Ludwigs University, Freiburg, Ger-many). Details about the system were reported elsewhere.15

Briefly, in contrast to traditional RP systems such as FusedDeposition Modelling, three-dimensional (3D) printing, Ste-reolitographie, Selective Laser Sintering, which mainly focuson a single mode of material processing, this system canaccommodate a much larger variety of synthetic and/ornatural biomaterials. A lay-down pattern of 0/90 was usedto form the honeycomb patterns of square with a single fillgap (FG) of 1 mm. For all the three polymers, porous sheetsmeasuring 40 � 40 mm were fabricated by using the systemwith a 0.5-mm diameter nozzle. The cartridge temperaturewas set at 70°C for PCL-PEG, 80°C for PCL-PEG-PEG, and100°C for PCL. The scaffolds were built on a piece of whiteprinter paper tapped down on the building platform, whichwas maintained at 20°C during the fabrication process. PCL-PEG and PCL-PEG-PCL were dispensed with an air pres-sure of 4.5 bars instead of 5 bars for PCL. For all the threepolymers, a speed of 50 mm/min in the x/y axis and a speedof 30 mm/min in the z axis were applied.

418 HUANG ET AL.

Cell culture studies

Human MSC isolation, culture, and seeding

Primary human mesenchymal precursor cells (MSC) wereisolated and cultured by using the same protocol as de-scribed previously.16 Three hundred thousand cells per scaf-fold (5 � 5 � 3 mm) were dynamically seeded at a densityof 1 � 105 cells/mL in six-well cell culture plates. Thescaffold/cells constructs were cultivated for 7 days undergentle dynamic culture conditions (10 rpm) on a platformshaker by using distinctive stem cell media (Mesencult, StemCell Technologies, Vancouver, BC) supplemented with 10%fetal bovine serum (FBS) and 1% penicillin/streptomycin.

Rat MSC isolation, culture, and seeding

Rat bone marrow derived stromal cells were isolated byflushing both femurs of male adults. Fisher rats (CharlesRiver Laboratories, Wilmington, MA) with 3 mL of saline-heparin solution (4:1, Pharmacia Upjohn). After centrifuga-tion at 1000 rpm for 10 min and washing twice with phos-phate-buffered saline (PBS), cells were cultured in DMEMlow glucose supplemented with 10% FBS and 1% penicillin/streptomycin. Nonadherent cells were removed with thefirst medium change at day 3. The bone marrow stromalcells were passaged once prior seeding on the scaffolds.

One day prior to cell seeding, 12 scaffolds of each polymermeasuring diameter 6 mm � 3 mm were cut out with adermal biobsy punch (Miltex Instrument Company, NY)and sterilized in 70% ethanol overnight. The ethanol wasremoved by centrifugation three times in changes of PBS for15 min with 1000 rpm. Noninduced progenitor cells of pas-sage 3 were detached by trypsin-ethylenediaminetetraaceticacid (EDTA) treatment, washed with PBS and resuspendedin media/fibrinogen solution in a ratio of 2:1, reaching aconcentration of 2 � 106 cells/mL. Twelve scaffolds of eachgroup were loaded with 40 mL fibrinogen/cell suspensionresulting in a seeding density of 3 � 106 rat mesenchymalprecursor cells (rMCP) per scaffold. The specimens werecultured under static conditions for 2 days. Thereafter, themedium was changed and the specimens were cultured inosteogenic medium I for 7 days and 5 weeks in osteogenicmedium II as described.16 During the culture period, super-natants were collected after specific time points, as indicatedprior to medium exchange and stored at �80°C.

Phase contrast light and scanning electronmicroscopy

Cell attachment, migration, proliferation, and extracellu-lar matrix production were daily examined by phase con-trast light microscopy for 2 and 3 weeks, respectively. Theadhesion and distribution of cells were studied via SEM.Specimens were fixed in 2.5% gluteraldehyde (Merck, Ger-many) for at least 4 h at 4°C. They were then dehydrated ina graded ethanol series of 30, 50, 90, and 100% for 5 min at

each grade, dried, and examined with a JEOL JSM-5800LVSEM at 15 kV.

Confocal laser microscopy (CLM)

Scaffold/cell constructs for CLM were prepared by stain-ing viable cells green with the fluorescent dye fluoresceindiacetate (FDA; Molecular Probes Inc., Eugene, OR). The 3Dcultures were incubated at 37°C with 2 g/mL FDA in PBS for15 min. After being rinsed twice in PBS, each sample wasplaced in a 1 mg/mL propidium iodide (PI) solution (Mo-lecular Probes) for 2 min at room temperature to stain deadcells red. The samples were then rinsed twice in PBS andviewed under a confocal laser microscope (Olympus IX70-HLSH100 Fluoview).

Alkaline phosphate assay

The amount of alkaline phosphates (n � 6) was quantifiedby measuring the conversion to p-nitrophenol from p-nitro-phenylphosphate according to the manufacturer’s protocol(ALP; Sigma 104; St. Louis, Mo). Supernatants of the cell-seeded scaffolds were taken at days 1, 3, 5, 10, 15, and 18 andfrozen at �80°C and thawed before analysis. All sampleswere performed in quadruplicates.

RESULTS

Synthesis and characterization

PCL homopolymer and its copolymers with PEGwere prepared by ring-opening polymerization of�-caprolactone in the presence of ethylene glycol orPEG. Table I summarizes the molecular characteristicsof these polymers. PCL-PEG and PCL-PEG-PCL wereprepared from monohydroxyl PEG (mPEG) with M� nof 5000 and dihydroxyl PEG with M� n of 8000, respec-tively, the initial [CL]/[EO] molar ratio being 3/1.This composition compromises the formation of highmolecular weights, appropriate PEG content, and thebioresorbability of PEG-rich degradation products. Infact, PEG with M� n lower than 30,000 Da can be ex-creted through kidney filtration.17 A [CL]/[EG] molarratio of 700/1 was used for the synthesis of PCL, and[CL]/[EO] ratios of 0.04/1 and 0.06/1 were used inOCL-PEG and OCL-PEG-OCL.

The [CL]/[EO] ratios of the resulted copolymerswere determined from the integration of the bandsdue to PCL blocks at 4.0 ppm and to PEG blocks at 3.6ppm on the 1H NMR spectra as previously de-scribed.13 The degree of polymerization (DP) was cal-culated according to the following equations:

DPPEG � (M� nPEG/44)

DEGRADATION AND CELL CULTURE STUDIES 419

DPPCL � DPPEG � ([CL]/[EO])

Figure 1 shows the X-ray diffraction patterns of PCL/PEG homo- and copolymers. PEG was characterizedby two main peaks at 9.4° and 11.5°, and PCL by anintense peak at 10.6° and a smaller one at 11.8°. PCL-PEG-PCL with long PCL chains exhibited the samecrystalline structure as PCL, indicating that the PEGsegment did not crystallize because of low PEG con-tent (�10 wt %). In contrast, OCL-PEG-OCL bearing

short OCL blocks retained the crystalline structure ofPEG. The diblock copolymers exhibited similar char-acteristics.

Thermal characteristics of the polymers in the formof powder were investigated by DSC in comparisonwith corresponding PEG. The melting enthalpy (�Hm)and melting temperature (Tm) data are shown in TableI. The �Hm values of mPEG5000 and PEG8000 were,respectively, 181 J/g and 154 J/g (i.e., much higherthan that of PCL). PCL-PEG and PCL-PEG-PCL pre-sented simular �Hm values as PCL in agreement withtheir low PEG content. On the other hand, OCL-PEGand OCL-PEG-OCL exhibited lower �Hm values thancorresponding PEG, showing that the crystallinity ofPEG can be greatly reduced by the presence of veryshort OCL chains. Tm values of PCL and PEG ho-mopolymers were very close (i.e., in the 68° to 69°Crange). PCL-PEG and PCL-PEG-PCL exhibitedslightly lower Tm (67° and 68°C) than PCL (69°C),whereas the Tm difference between OCL/PEG copol-ymers and parent PEGs was larger.

The behavior of OCL-PEG and OCL-PEG-OCL inaqueous solutions was examined by micelle forma-tion. Two milligrams of each polymer were dissolvedin 5 mL of 0.13 M, pH 7.4 phosphate buffer in thepresence of a water-insoluble dye, namely yellow OB,which is known to dissolve in the hydrophobic core ofpolymeric micelles or aggregates.18 After sonicationand centrifugation, the solutions became colored, in-dicating micelle formation. This finding showed thatOCL-PEG and OCL-PEG-OCL block copolymers ex-hibited an amphiphilic behavior.

Hydrolytic degradation

Visual aspect

Initially, the various films appeared homogeneousand opaque. Beyond 18 weeks, PCL-PEG and PCL-PEG-PCL became increasingly whitish and brittle,

TABLE IMolecular Characteristics of PCL and Its Block Copolymers With PEG

Polymer Initiator

[CL]/[EO] [CL]/[EO]a

DPPEGb DPPCL

b M� nc M� w/M� n

c�Hm(J/g)d

Tm(°C)dIn feeds In Products

PCL EG 700/1 — — — 50,000 1.7 56 69PCL-PEG mPEG5000 3/1 3.9/1 114 445 33,000 1.6 62 67PCL-PEG-PCL PEG8000 3/1 3.7/1 182 672 30,000 1.8 48 68OCL-PEG mPEG5000 0.04/1 0.03/1 114 3 2,300 1.4 117 67OCL-PEG-OCL PEG8000 0.06/1 0.04/1 182 7 2,100 1.5 116 60

aThe [CL]/[EO] molar ratio was determined by 1H NMR.bDPPEG � M� n,PEG/44, DPPCL � DPPEG � ([CL]/[EO]).cData obtained by SEC with respect to polystyrene standards.dThermal properties were investigated by DSC.

Figure 1. X-ray diffractograms of (a) PEG8000, (b) OCL-PEG-OCL, (c) PCL-PEG-PCL, and (d) PCL.

420 HUANG ET AL.

whereas PCL remained rigid. On the other hand, allthe polymers retained their initial geometrical shapesduring degradation.

Water absorption and weight loss

Water absorption reflects the bulk hydrophilicity ofa polymer. PCL absorbed only 0.5% after 60 weeks[Fig. 2(a)], in agreement with its high crystallinity andhydrophobicity. In contrast, both PCL-PEG and PCL-PEG-PCL absorbed about 5% of water within 2 weeks.Afterward, water absorption data showed some fluc-tuation and reached 8% for PCL-PEG-PCL and 6% forPCL-PEG after 60 weeks.

Weight loss of the samples reflects the release ofsoluble oligomers formed during degradation. No sig-nificant weight loss was detected for PCL during thewhole degradation period. In the copolymers, weightloss was detected after 9 weeks [Fig. 2(b)]. Beyond, itincreased slowly to reach 3.3% for PCL-PEG and 7.5%for PCL-PEG-PCL at week 60. The latter showedhigher weight loss than the former due to the highersolubility of PEG8000-rich species, in agreement withwater absorption data. In fact, water was retainedwithin the PEG segments and in the voids left bydiffusion of PEG-rich degradation products.

Molecular weight (MW)

SEC was used to monitor the evolution of MW andMW distributions. PCL presented initially an M� n �50,000 and a rather narrow MW distribution withM� w/M� n � 1.7. As the degradation proceeded, M� ndecreased to 38,000 after 18 weeks, and to 33,000 after45 weeks. The same trend was found for the copoly-mers. M� n of PCL-PEG decreased from initial 33,000 to30,000 and to 16,000, whereas M� n of PCL-PEG-PCLdecreased from initial 30,000 to 18,000 and 14,000 after18 and 45 weeks, respectively. In all cases, the MWdistribution remained monomodal (Fig. 3), indicatinghomogeneous hydrolysis.

Composition

Compositional changes of PCL-PEG and PCL-PEG-PCL during degradation were monitored by 1H NMR.The composition remained unchanged during the first9 weeks. At week 18, the [CL]/[EO] ratio increased

Figure 3. SEC chromatograms of (a) PCL, (b) PCL-PCL, (c)PCL-PEG-PCL after (—) 0 and (- - -) 45 weeks of degrada-tion.

Figure 2. (a) Water absorption and (b) weight loss profilesof PCL (•), PCL-PEG (E), and PCL-PEG-PCL (‚) duringdegradation

DEGRADATION AND CELL CULTURE STUDIES 421

from 3.9 to 4.3 for PCL-PEG and from 3.7 to 4.6 forPCL-PEG-PCL. Beyond, it continued to increase andreached 7.9 for PCL-PEG and 10.2 for PCL-PEG-PCLat week 60. The latter exhibited larger compositionalchanges than the former, in agreement with weightloss data. Therefore, the released soluble oligomerscontained large amount of PEG. In other words, theremaining material was enriched in PCL component.

Thermal properties

Thermal property changes were followed by DSC asshown in Table II. A slight Tm increase was detectedfor PCL, PCL-PEG, and PCL-PEG-PCL during degra-dation. �Hm of PCL increased almost constantly, inagreement with the continued crystallization. In fact,PCL could crystallize at 37°C, which was much higherthan its Tg (�65°C). �Hm of PCL-PEG and PCL-PEG-PCL exhibited a slight decrease or fluctuation, whichcould be related to water absorption and release ofPEG-rich species. At the later stages, however, anincrease in �Hm was detected for both copolymers.

Release of water-soluble oligomers

The release of water-soluble species in the aqueousphase was determined by CZE according to litera-ture.19,20 6-hydroxyhexanoate (6-HH) was detected atweek 2 for the copolymers and at week 5 in the PCL.The amounts of 6-HH remained at very low levelscompared to the weights of the films (below 1.0 wt %).It could be assigned to residual �-caprolactone initiallypresent within the matrix. There were no dimer, tri-mer, or higher oligomers detected.

The test of micelle formation in the aqueous phasewas performed in the presence of yellow OB. Aftersonication and centrifugation, the solutions remaineduncolored, indicating the concentration of amphiphilicdegradation products was below the critical micelliza-

tion concentration (CMC) during the whole degrada-tion period.

Scaffold fabrication

Using the 3D dispensing RP process, the pore mor-phology can be varied by changing the lay-down pat-tern used (i.e., by changing the raster angle of depo-sition), the width of the deposited roads, and/or thespacing between the roads. The pore openings facingthe z-direction are formed in between the intercross-ing of roads and are determined by user-defined pa-rameter settings as mentioned above. However, thepore openings facing both the x- and y-directions areformed from voids created by the stacking of materiallayers and hence, their sizes are determined by thediameter of the nozzle and the melt viscosity of thepolymer. Because the process uses the continuousdeposition of melted material to form each layer [Fig.4(a)], pore openings in both the x- and y-directions arepartially occluded as the combination of polymerproperties and processing parameters are not opti-mized, in contrast to commercial RP systems such asFDM and so forth [Fig. 4(b)].

Cell culture studies

Human MSC

Dynamically seeded hMSC attaching to the PCL-PEG and PCL-PEG-PCL scaffolds appeared initiallyround as observed via phase contrast light microscopy(data not shown). After 3 days in culture, the cellsrevealed distinct morphological changes whilespreading on the bars and struts of the scaffold surfaceas well as bridging of adjacent bars as shown by CLM

TABLE IIThermal Property Changes of PCL, PCL-PEG, and PCL-PEG-PCL With Degradation

PolymerDegradation

Time(weeks)

PCL PCL-PEG PCL-PEG-PCL

�Hm (J/g) Tm (°C) �Hm (J/g) Tm (°C) �Hm (J/g) Tm (°C)

0 47 66 60 64 55 632 51 67 52 66 47 665 49 68 46 67 53 669 nd nd 52 67 51 67

18 68 67 52 67 54 6730 70 67 50 68 55 6845 72 66 (68)* 56 68 (69)* 65 6860 65 70 64 68 74 67 (69)*

*Double melting peak.

422 HUANG ET AL.

(Fig. 5). In addition, focal clusters of viable round cellswere observed within the pores of all three scaffoldgroups, which appeared to be loosely attached to thesurface of a bar/strut. As indicated by the confocalimages using the live/dead stain FDA-PI, most cellswere viable at day 3.

An increase in apoptosis was observed with increas-ing culture time due to the fact that nutrients could notreach the cells inside the clusters (Fig. 5). The 3D cellclusters were not visible in the SEM images due to thefixation and specimen processing resulting in a de-tachment of the cell-conglomerates. The SEM micro-graphs (Fig. 6) show that most bars and struts of bothscaffold types were covered by a dense cell-sheet.From qualitative assessment, cellular attachment andproliferation appeared to be higher on the PCL-PEGscaffold than on the PCL-PEG-PCL.

Rat MSC

During the first 2 days of culture, the cells remainedround within the fibrinogen gel and revealed a ho-mogenous distribution within all three scaffoldgroups (Fig. 7). Beyond day 3, the cells began tostretch and proliferated within the biomimetic hydro-gel and showed a fibroblast-like phenotype for allthree groups (Fig. 7). CLM (Fig. 8, upper row) andSEM (Fig. 8, lower row) revealed the adherence ofmesenchymal progenitors onto the PCL-PEG, PCL-

PEG-PCL, and PCL fibers and, after 2 weeks in cul-ture, bridging the pores of both scaffolds in combina-tion with the production of extra cellular matrix.Henceforth, phase contrast microscopy and SEMshowed that after 3 weeks the entire architecture ofboth scaffold groups was filled with cells and extra-cellular matrix. This was in accordance with CLMimages. Qualitative examination of cells via routineCLM did reveal that a large number of cells stayedactive for the entire culture period, which was inconformity with the alkaline phosphatase (ALP) spe-cific activity results (Fig. 9).

Based on the imaging data, a significant differencebetween PCL-PEG and PCL-PEG-PCL in respect to theproliferation pattern could not be detected. The ALPtest (Fig. 9) revealed almost linear increase of cellmetabolism up to day 12 for both block copolymersuntil a constant metabolic level was reached, followedby a decrease in ALP levels up to day 18. This was inaccordance with image analysis, which revealed thatafter prolonged cultivation of scaffold/cell constructs,by day 28, FDA/PI staining still showed vital cellsinside the honeycomb scaffold architecture (data notshown).

Biochemical analysis of osteogenic differentiation ofthe rMSC using the ALP ELISA assay indicated thatALP specific activity was higher in the copolymersthan in the pure PCL scaffolds. Cell seeded PCL-PEGexpressed the highest values over the whole time

Figure 4. (a–e) Scanning electron micrographs of the PCL-PEG and PCL-PEG-PCL scaffolds and schematic drawing of thefilament lay-down pattern during the extrusion process. (a) and (b) PCL-PEG scaffold in a top and side view. (c) and (d)PCL-PEG-PCL scaffolds. (Note the smaller side width due to the plastic deformity of the freshly extruded filament. Thetriblock-scaffolds have a higher surface porosity compared to the diblock scaffolds.) [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]

DEGRADATION AND CELL CULTURE STUDIES 423

course with a peak at day 12 followed by a decreasingtendency up to day 18. PCL-PEG-PCL seeded scaf-folds showed a peak in ALP values at day 12, followedby decreasing values. The cells on the PCL scaffoldsexpressed the lowest amount of ALP with a continu-ously decreasing tendency up to day 18.

DISCUSSION

The hydrolytic degradation of aliphatic polyesters isa very complex process involving various diffusion-reaction phenomena such as water absorption, ester

bond cleavage, diffusion, and solubilization of solublespecies.3,4 Pitt et al.3 studied the in vivo degradation ofPCL films and capsules. The authors suggested thatdegradation consists of two stages. The first stagestarts with random hydrolytic ester cleavage autocata-lyzed by the carboxyl endgroups, the MW decreasingcontinuously. The second stage is characterized by adecrease in the rate of chain scission and the onset ofweight loss due to the formation of fragments smallenough to diffuse out of the polymer bulk. In thepresent study, water absorption of PCL film remainedvery low due to its high crystallinity and hydropho-bicity. The SEC chromatogram of PCL was mono-modal and shifted continuously to lower MW, indi-cating a homogeneous hydrolysis. Weight loss andmonomer formation remained negligible because thefragments were not small enough to be soluble inwater and diffuse out of the polymer bulk.

PCL-PEG and PCL-PEG-PCL exhibited a higher hy-drophilicity because of the presence of PEG blocks.The SEC chromatograms were monomodal andshifted to lower MW with degradation, in agreementwith a homogeneous hydrolysis. Weight loss andcompositional changes were detected beyond 9 weeks.1H NMR analysis revealed that large amounts of PEG-rich species were released out of the bulk duringdegradation. 6-HH was detected from the very begin-ning in the buffer solution and fluctuated at a very lowlevel, indicating the presence of residual �-caprolac-tone within the matrix. Over the whole degradationperiod, no micelles were formed in the aqueous me-dium, showing that the concentration of soluble oli-gomers remained below the CMC.

To our knowledge, the use of SFF fabricated scaf-folds based on PCL/PEG copolymers has not beenextensively explored in the tissue-engineering litera-ture. The initial cell culture studies were performedwith the objective of investigating cell adhesion, pro-liferation, and differentiation on PCL-PEG and PCL-PEG-PCL in comparison with PCL. The characteriza-tion of the various surfaces revealed increasedhydrophilicity for the copolymers as demonstrated bycontact angle measurements (data not shown), inagreement with water absorption data [Fig. 2(a)]. Thiswould allow increased perfusion of cells and nutri-ents, thus increasing efficiency during cell seeding.One limitation of this study was that wettabilitychanges were measured only on two-dimensional sur-faces. Nevertheless, the results were considered asrelevant because the films for wettability tests wereprepared via melt molding. Contact angle measure-ments on the highly porous structure of scaffolds wereattempted, but the results were not reproducible.

The melt extrusion process on the RP machine re-sulted in a rough polymer surface and internal matrixpitting as shown by SEM (Fig. 4). Established RPsystems mainly focus on a single mode of material

Figure 5. (a–f) A series of confocal laser microscopy imagesof the dynamically seeded scaffolds after 1 and after 7 days inculture. (a) PCL-PEG scaffold and (b) a PCL-PEG-PCL scaffoldafter 1 day of culture. The diblock scaffold has a higher numberof cells attached to the bars than the triblock scaffold. Theviable cells are stained with the green dye FDA, and nuclei ofdead cells are indicated by red PI stain. The cell clusters in thepores show a high uptake of the FDA stain, indicating that themajority is alive. After 7 days in culture, the cells in the clusterare partially dead for both scaffolds [Fig. 5(c) and (d): bothoriginal magnification � 100]. (e) and (f) show a higher mag-nification (�200) for both cell seeded scaffolds. Note there is aincreased rate of dead cells (red PI stain) in the triblock scaf-folds. [Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]

424 HUANG ET AL.

processing. The 3D plotter was designed to give thetissue engineer the possibility to process a large vari-ety of natural and synthetic polymers. However, thisadvantage makes it more difficult to optimize thesetup processing conditions and finally manufactur-ing of specific polymeric scaffolds. A number ofgroups have reported low seeding efficiency resultswhen pipetting cells with small volume of superna-tants into 3D scaffolds followed by addition of culturemedia at different time intervals.21,22 Based on thisbackground, dynamic cell seeding was studied toovercome the low seeding efficiency.23–26 Scaffoldsused in the present study with a honeycomb andchannel-like architecture do have a low surface to

volume ratio. As a consequence, dynamic seeding re-sulted in the formation of large cell clumps inside thepores with focal contact point to the scaffold surface.Thus, dynamic seeding is a critical issue for this typeof scaffold architectures, strong cell to cell contactbeing characteristic for hMSC.27–29

However, enhanced cell attachment on PCL-PEG sur-faces observed by CLM and SEM could also have pro-moted cell proliferation. Through a phenomenon knownas anchorage dependence, increased avidity of cell bind-ing stimulates cells to enter the S phase of mitosis. There-fore, increased cell proliferation could have resultedfrom a combination of cytokine stimulation and higheranchorage dependence of PCL-PEG surfaces.

Figure 6. Scanning electron microscopy images of the cross-sectional side view of the PCL-PEG (a) and the PCL-PEG-PCL(b) scaffolds after 7 days in culture. The entire surface of the bars and struts is covered by a dense cell-ECM matrix. Cellsbridging adjacent bars are present.

Figure 7. Transmitted phase contrast light microscopy images of PCL (a and b), PCL-PEG (c and d), and PCL-PEG-PCL (eand f) seeded with rat bone marrow stromal cells (rMSC) after 1 (upper row) and 2 weeks (lower row) in culture. The cellscontinued to proliferate on all three matrices, leading to almost complete obliteration of the porous space of the scaffolds. Cellattachment and proliferation were increased on the PCL and PCL-PEG scaffolds compared to the PCL-PEG-PCL scaffolds (allimages magnification �10). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

DEGRADATION AND CELL CULTURE STUDIES 425

One of the issues to overcome the low seeding effi-ciency is to use a biomimetic cell carrier, which mightcontain not only a number of binding proteins (fi-

bronectin, etc.), but also allow the homogenous deliv-ery of cells into synthetic polymer framework.21 Abiomimetic hydrogel was used as cell carrier in the ratMSC culture study and almost 100% seeding effi-ciency was achieved.

CLM, SEM, and phase contrast light microscopyrevealed increased cell density over time not only atthe outer surface but also throughout the entire scaf-fold architecture. This finding bears significance intissue engineering in which in vitro neo-tissue forma-tion (cells ECM) is desirable for achieving homog-enous tissue formation in combination with vascular-ization in vivo.6 Our findings in this study suggestedneo-tissue formation throughout the polymeric scaf-fold architecture.

With increased culture time, ALP specific activitysuggests that rBMPC did start to go down the osteo-blastic pathway. However, there was no evidence ofstrong mineralization during the end of the cultureperiod. Because cell differentiation precedes mineral-ization, further studies of longer duration are neededto evaluate the effect of PCL/PEG copolymers onmineralizing the neo-tissue in vitro.

The specific mechanism that promotes or blocks cellattachment, proliferation, and differentiation on PEG-

Figure 8. Confocal laser microscopy images and corresponding scanning electron micrographs from the rMSC seededscaffolds after 3 weeks in culture (a and b: PCL; c and d: PCL-PEG; e and f: PCL-PEG-PCL). The porous space of the PCL andthe diblock scaffolds is nearly completely filled with viable (FDA, green stained; original magnification �10) cells. The cellsanchored with multiple lamellipodia to the fibrin coated scaffolds like roots of a tree, bridging adjacent bars. Due to the SEMprocessing, most cells are detached from the scaffold pores. [Color figure can be viewed in the online issue, which is availableat www.interscience.wiley.com.]

Figure 9. Alkaline phosphatase-specific activity of the ratbone marrow stromal cells on the three matrices. ALP activ-ity was significantly higher on the PCL-PEG scaffolds ascompared to the other two scaffold types. The ALP activityincreases for the PCL-PEG and PCL-PEG-PCL scaffold up today 12, indicating osteogenic differentiation of the precursorcells due to the media supplements followed by a slowdecrease up to day 18, indicating the second phase of osteo-genic lineage progression with remodeling of the subpopu-lation and increased apoptosis.

426 HUANG ET AL.

containing copolymers is not fully understood yet.However, it is known that PEG incorporation resultsin chemical and physical changes on the copolymersurface. Either of these changes could influence cellfunction by altering the deposition of biochemicalmoieties on the surface. For instance, surface changescould potentially influence the adsorption and foldingof proteins on the surface, which would impact cellu-lar attachment and function subsequently.

Scaffolds based on PCL/PEG copolymers have notbeen extensively explored in the field of regenerativemedicine. However, this type of biodegradable poly-mers allows scaffolds for specific tissue-engineeringapplications to be designed and fabricated. With thispotential in mind, future studies will investigate theeffect of PCL/PEG copolymers on other cell types invitro and in vivo.

Cell culture studies reported in this paper were funded bya National University of Singapore Young InvestigatorAward, WBS No. R-397-000-003-712. The authors would liketo thank Prof. Muellhaupt and Dr. Rudiger landers for theirsupport in fabricating the scaffolds.

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