Biomaterials 25 (2004) 1087–1093

ARTICLE IN PRESS

*Correspondin

1647 (S.R. Bha

(G.N. Tew).

E-mail addres

tew@mail.pse.um

0142-9612/$ - see

doi:10.1016/S014

Rheological studies of PLLA–PEO–PLLA triblock copolymerhydrogels

Khaled A. Aamera, Heidi Sardinhab, Surita R. Bhatiab,*, Gregory N. Tewa,*aDepartment of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003, USA

bDepartment of Chemical Engineering, University of Massachusetts, Amherst, MA 01003, USA

Received 2 June 2003; accepted 25 July 2003

Abstract

We report detailed rheological data on aqueous gels formed from triblock copolymers of l-lactide and ethylene oxide including

the dependence of the viscoelastic moduli on frequency and applied stress of these systems for the first time. We are able to create

strong gels with elastic moduli greater than 10,000 Pa, which is an order of magnitude higher than previously achieved with related

biocompatible physically associated gels of similar chemistry. Moreover, the value of the elastic modulus strongly depends on PLLA

block length, offering a mechanism to control the mechanical properties as desired for particular applications. At the gel point, we

observe scaling that is characteristic of a percolated network, G0B G0 0BoD, but with an exponent that is lower than predicted by

percolation, D=0.36. Our results have implications for the design of new materials for soft tissue engineering, where native tissueshave moduli in the kPa range.

r 2003 Elsevier Ltd. All rights reserved.

Keywords: Polylactic acid; Polyethylene oxide; Triblock; Elasticity; Hydrogel; Mechanical properties

1. Introduction

Amphiphilic polymers have received considerableattention as potential biomaterials in the last decade[1–5]. In particular, materials capable of forminghydrogels remain an active area of research due toapplications in drug delivery, tissue engineering andother biomedical devices [3,4]. Hydrogels are attractivefor a variety of reasons including less invasive medicalprocedures and the fact that these materials most closelymimic natural tissues in water content, interfacialtension, and mechanical properties (soft and rubbery)[5]. Triblock copolymers of poly (l-lactide) (PLLA) andpoly (ethylene oxide) (PEO) have been studied exten-sively since the initial report by Cohn [6] because of thebiodegradability of the polyester and the biocompat-ibility of PEO [7–21]. In addition, these materials areknown to form hydrogels of varying weight %,

g authors. Tel.: +1-413-545-0096; fax: +1-413-545-

tia); Tel.: +1-413-577-1612; fax: +1-413-545-0082

ses: sbhatia@ecs.umass.edu (S.R. Bhatia),

ass.edu (G.N. Tew).

front matter r 2003 Elsevier Ltd. All rights reserved.

2-9612(03)00632-X

depending on the exact architecture, with transitionsnear body temperature. Most studies have focused onthe sol–gel transition temperatures by vial inversion andthus do not provide detailed mechanical properties[16,22–26]. In the study presented here, we synthesizedPLLA–PEO–PLLA polymers containing large PEO andvarying PLLA blocks which were characterized in thegel phase by mechanical rheology. Specifically, ourdesire is to find strong physically cross-linked hydrogelsfrom polyester ether materials for potential applicationin biomedical devices.Amphiphilic copolymers composed of PLA and PEO

have been prepared with PEO end- or mid-blocks andcharacterized as hydrogels [16,22–26] by vial inversionmethods which require elastic modulus greater than65 Pascals (Pa) [27]. For the triblock architecture ofinterest here, PLA–PEO–PLA, previous reports haveformed gels with polymers composed of lactic andglycolic acid, PLGAx–PEO1000–PLGAx, in which x, thedegree of polymerization (DP), was varied both in blocklength and lactide to glycolide ratio [22]. These materialsexhibited sol–gel transitions between 10�C and 23�Cand gel–sol transitions from 27�C to 48�C depending onconcentration and x [22]. The same group showed

ARTICLE IN PRESSK.A. Aamer et al. / Biomaterials 25 (2004) 1087–10931088

that polymers composed of d, l lactide formed gelswith a sharper gel–sol transition compared to PLGApolymers [23]. More recently, mechanical rheology onPMG1400–PEO1450–PMG1400 (PMG-poly(d,l-3-methyl-glycolide)) showed the elastic modulus of a 27wt%sample is less than 500Pa [28]. Further, Kimura and co-workers reported hydrogel formation from stereocom-plexed PLLA1300–PEO4600–PLLA1300 and PDLA1090–PEO4600–PDLA1090 in which 10wt% solutions had anelastic modulus up to 1000 Pa at 37�C [29]. Solutions ofeither polymer independently at 10wt% did not form ahydrogel. Thus, a relatively complicated system wasrequired to obtain favorable mechanical properties. Inaddition, all rheological data reported in these studieswere taken at a single frequency and strain, so thedetailed dependence of modulus on frequency andapplied strain is completely unknown.The polymers described herein are composed solely of

l-lactide and contain PEO blocks that are larger thanthose utilized in previous studies (MW=8900Da). Wereport values for the elastic modulus (G0) and lossmodulus (G00) over a range of frequency and appliedstress, allowing for a more complete characterization ofthe rheological properties. Moreover, we focus onsystems with hydrophobic blocks that have a lowermolecular weight than the PEO block, which representsa significant difference between the majority of PLA–PEO–PLA systems reported in the literature. Thisenables us to create physically associated gels that areanalogous to reversible network gels formed fromtelechelic hydrophobically modified polymers. As dis-cussed further below, this leads to gels with elasticmoduli that are significantly higher than previouslyreported.

2. Experimental section

2.1. Materials

l-lactide (Aldrich) was purified by recrystallization indry ethylacetate and by sublimation prior topolymerization. The a, o dihydroxy polyethyleneglycol macroinitiator with molecular weight 8900(PEG 8K, Aldrich) was dried at room temperatureunder vacuum prior to polymerization. Stannous (II) 2-ethyl hexanoate (Alfa Aesar) was used without furtherpurification.

2.2. Synthesis of PLLA–PEO–PLLA triblock copolymer

PLLA–PEO–PLLA triblock copolymers were synthe-sized by bulk polymerization. PEO, was introduced intoa dried polymerization tube. The tube was purged withnitrogen, and placed in an oil bath at 150�C. Stannous(II) 2-ethyl hexanoate was introduced under nitrogen to

the molten PEO and stirred for 10min followed byaddition of l-lactide to the macroinitiator/catalyst melt.The polymerization was carried out at 150�C for 24 hwith stirring, after which it was quenched by methanol.The product was dissolved in tetrahydrofuran andprecipitated in n-hexane. The process of dissolution/reprecipitation was carried out three more times. Thecopolymer was dried under vacuum at room tempera-ture for 2 days.

2.3. Sample preparation and instrumentation

The copolymers polydispersity are measured versuspolyethylene oxide standards using GPC (HP 1050series, a HP 1047A differential refractometer, and threePLgel columns (5 mm 50 (A, two 5 mm MIXED-D) indimethylformamide as eluting solvent at a rate of 0.5ml/min rate at room temperature. The copolymer composi-tions are determined by 1H NMR (Bruker, DPX300,300MHz spectrometer, d-chloroform).Gels were prepared by slow addition of dried polymer

sample to a fixed volume of DI water (15ml) followedby stirring and heating. Gels were then transferred to aBohlin CVO rheometer for oscillatory measurements. Acone-and-plate geometry with a 4� cone, 40mmdiameter plate, and 150mm gap was used for allexperiments on hydrogels. For liquid samples with alow viscosity, a couette geometry was used. Stressamplitude sweeps were performed to ensure thatsubsequent data was collected in the linear viscoelasticregime. Frequency sweeps were performed at a constantstress (0.1–2.0 Pa, depending on the sample) in thefrequency range 0.01–100Hz. At high frequencies, aresonant frequency of the rheometer motor wasobserved; thus, data are reported up to a frequency ofapproximately 10Hz, again depending on the particularsample.

3. Results and discussion

3.1. Block copolymer synthesis

The copolymers shown in Table 1 were prepared byring-opening polymerization of l-lactide at 150�C in thebulk using stannous (II) 2-ethylhexanoate as catalyst.This method is known to limit racemization of thestereocenter and produce polymers of significant mole-cular weight and narrow polydispersity [7]. The macro-initiator, PEO, has molecular weight (MW) of 8900Daand four different polymers were prepared with increas-ing PLLA block lengths. These lengths varied from atotal DP of 26 to 74 so that the total lactide compositionis always smaller than PEO. 1H-NMR integration wasused to establish theMw for PLLA blocks as opposed toGPC standards [10,11]. In all cases, the polymerization

ARTICLE IN PRESS

Table 1

Molecular weight characteristics of PLLA–PEO–PLLA triblock copolymers

OHO

H202

+O

O

O

O Poly(LLA-EG-LLA)

OO

O

O

HO

Oy y150oC/Bulk polymerization

Sn(Oct) 2 H202

Sample MnPEGa MnPLLA

b MTotal MWDc Total DPPLLAb Wt%PEO Wt%PLLA

1 8900 1872 10,772 1.31 26 82.6 17.4

2 8900 3744 12,644 1.24 52 70.4 29.4

3 8900 4320 13,220 1.18 60 67.3 32.7

4 8900 5328 14,228 1.20 74 62.6 37.4

aDetermined by MALDI TOF and GPC.bDetermined by 1H NMR.cDetermined by GPC.

100

1000

10000

100000

0.01 0.1 1 10

Frequency (Hz)

G',

G"

(Pa)

G'G"

Fig. 1. Elastic modulus (filled diamonds) and viscous modulus (open

squares) as a function of frequency for a gel of sample 4 at 16wt%

polymer and T=25�C.

0.1

1

10

100

1000

10000

100000

0.01 0.1 1 10 100

Frequency (Hz)

G',

G*

(Pa)

G*, 1, 25 CG', 2, 25 CG', 3, 25 CG', 4, 25 CG', 2, 37 CG', 3, 37 CG', 4, 37 C

Fig. 2. Elastic moduli versus frequency for gels formed from a series

with increasing PLLA block length, samples 2, 3, and 4 at

concentrations of 20, 16, and 16wt%, respectively; at 25�C (filled

symbols) and 37�C (open symbols). For comparison, the complex

modulus of a 20wt% solution of sample 1 is also shown.

K.A. Aamer et al. / Biomaterials 25 (2004) 1087–1093 1089

was not run to high conversion since this broadens themolecular weight distribution.

3.2. Rheology

All samples were characterized by dynamic mechan-ical rheology. Solutions of the triblock with the smallesthydrophobes (1) did not gel up to concentrations of20wt% polymer, while the remaining three triblocksformed gels at moderate concentrations (16–20wt%).Fig. 1 shows G0 and G00 vs. frequency for 16wt% 4 at25�C. The gel displays a high degree of elasticity, with G0

only weakly dependent on frequency and greater thanG00 over the entire frequency range. Note thatG0X10,000 Pa for this system, which is an order of

magnitude higher than the stereocomplexed systemsstudied by Kimura and co-workers [29]. Biomaterials

with moduli in the kPa range are of widespread interestsince many native tissues have moduli in this range,although most have nonlinear response to strain. Forexample, human nasal cartilage (234727 kPa) [30,31],bovine articular cartilage (990750 kPa) [30,31], pigthoracic aorta (43.2715 kPa) [32], pig adventitial layer(4.7271.7 kPa) [32], right lobe of human liver(270710 kPa) [33], canine kidney cortex and medulla(B10 kPa) [34], and nucleus pulposus and eye lens(B103 Pa) [34] have moduli in this range. Moreover, forscaffolding applications, it is often desirable to ‘‘match’’mechanical properties of the polymer matrix to those ofthe surrounding tissue [35].Fig. 2 compares the elastic modulus of systems

containing 2, 3, and 4 at concentrations of 20, 16, and16wt%, respectively, at 25�C (filled symbols) and 37�C(open symbols). These represent a series in the length of

ARTICLE IN PRESS

1

10

100

1000

0.01 0.1 1 10

Frequency (Hz)

Ela

stic

Mo

dulu

s (P

a)

25 C

37 C

40 C

50 C

Fig. 3. Elastic modulus versus frequency for a gel with 20wt% of

sample 2 as a function of temperature. Note the non-monotonic

dependence of G0 on temperature for this sample.

1000

10000

100000

0.01 0.1 1 10

Frequency (Hz)

Ela

stic

Mo

dulu

s (P

a)

25 C 31 C37 C 49 C61 C 70 C

Fig. 4. Elastic modulus versus frequency for a gel with 16wt% of

sample 4 as a function of temperature.

1

10

100

1000

0.01 0.1 1 10

Frequency (Hz)

G',

G"

(Pa)

G'G"

1

10

100

1000

0.01 1

T = 50 C

T = 37 C

Fig. 5. Elastic modulus (filled diamonds) and viscous modulus (open

squares) versus frequency for a 20wt% gel of sample 2 at T=37�C and

(inset) T=50�C.

K.A. Aamer et al. / Biomaterials 25 (2004) 1087–10931090

the hydrophobe, with 2 having the shortest PLLAblocks. Although the gel of 2 was prepared in 20wt%, asopposed to 16wt% for the other samples, it still forms aweaker gel. It is clear from these results that thehydrophobe length or DP has a pronounced effect onelastic modulus. The only difference between thesepolymers is the total DP of the PLLA segments, whichare 52, 60, and 74, respectively. As the length of thehydrophobic block is increased from 26 (half of 52 sincethere are two PLLA ends) to >30, gel strength improvesby more than two orders of magnitude fromB100 Pa to10,000 Pa even though the percentage of polymer in thegel is decreased. For comparison, the complex modulusG� of a 20wt% solution of 1 at 25�C is also shown inFig. 2. This sample, with hydrophobic block lengthsof 13, forms a viscoelastic liquid with G�o1 Pa.Qualitatively similar trends have been observed for thehigh-frequency limit of G0 for PEO containing alkylhydrophobe end-caps [36]. However, the dependence ofG0 on hydrophobe length is much weaker for these alkyl-capped systems than in our PLLA–PEO–PLLA gels,while the high-frequency elastic moduli of gels offluoroalkyl-capped PEO were found to be insensitiveto the hydrophobe length [37,38]. Thus, the strongdependence of G0 on the PLLA block length should offera straightforward way to tune the rheological responseof our gels for specific bioapplications.The elastic modulus at 37�C is also shown on Fig. 2

for these gels. All three gels display a decrease in G0 withtemperature; however, with a much larger decrease for 2than either 3 or 4. This decrease in G0 with increasingtemperature is common for gels composed of PLA–PEO–PLA molecules [23]. It is important to note that,despite this decrease, the value of G0 for 4 remains highat physiological temperatures (roughly 10,000 Pa), andthat both 3 and 4 are still elastic gels with G0 nearlyindependent of frequency.Figs. 3 and 4 show the influence of temperature on G0

for samples 2 and 4. Interestingly, G0 for sample 2

decreases with temperature as expected; however, near40�C the sample undergoes a transition toward increas-ing G0. At 50�C, the elastic modulus is even more linearwith temperature and significantly higher at lowfrequency than found at 37�C. The physical andstructural properties of the gel responsible for thesechanges are not understood at this time. However, it ispossible that at 25�C, the initial measurement, thehydrogel is composed of slightly hydrated micelles, butas the temperature is increased near 40�C, these micellesbegin to melt and destabilize (Tg of PLLA is approxi-mately 52�C and should be lowered by the presence ofwater as a weak plasticizer). Then, as the temperature isincreased further, the PLA domains may dehydrate in amanner similar to that observed for the PPO segmentsof Pluronics [39]. The observed behavior is consistentwith this desolvation transition but more work must be

performed to prove or disprove this assumption.Alternatively, 4 behaves in a more expected manner(Fig. 4) with a steady decrease in G0 as the temperature is

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1

10

100

1000

10000

100000

0.00001 0.0001 0.001 0.01 0.1

Strain

Ela

stic

Mod

ulus

(Pa)

2

3

41

10

100

1000

10000

100000

0.01 0.1 1 10 100

Stress (Pa)

Ela

stic

Mod

ulus

(Pa)

2

3

4

(a) (b)

Fig. 6. Elastic modulus at a fixed frequency of 1.0Hz as a function of (a) strain and (b) stress for samples 2, 3, and 4 at concentrations of 20, 16, and

16wt%, respectively, and 25�C.

K.A. Aamer et al. / Biomaterials 25 (2004) 1087–1093 1091

increased and this would be expected because water isless likely to hydrate larger PLLA blocks. However, at70�C, the data shows a slight increase in modulusconsistent with PEO dehydration.Sol–gel transitions can be defined as G0>G00, and

Fig. 5 shows that for 2 at 37�C G0EG00 for the entirefrequency range. Interestingly, the observed increasein G0 at 50�C for sample 2 is confirmed in the inset ofFig. 5, where G0 is much larger than G00. The frequency-dependent G0 observed for sample 2 at 25�C and 37�C,as opposed to the frequency-independent behaviorobserved at 50�C, may indicate the presence of a slowrelaxation mechanism at lower temperatures.The data on 2 at the gel point (37�C) can be fit to a

power law scaling of the form G0B G00BoD [40]. Here, ois frequency and D is an exponent whose value at the gelpoint can be predicted to lie in the range 0.67–1.0 fornetworked gels through classical mean-field considera-tions [41], electrical network analogies [42], and percola-tion theory [43,44]. Modified percolation models thataccount for elasticity of the network backbone [45,46]and excluded volume effects [47] have yielded predic-tions in the range 0–1.0. Fitting the data shown in Fig. 5to a power law gives D=0.36. Although it is impossibleto draw any definite conclusions about the structure ofthe gel from this value, similar values have been reportedfor nonstoichiometric chemically crosslinked polymericgels with an excess of crosslinker present, and forchemically crosslinked gels with chains above theentanglement molecular weight [48]. By contrast,experiments on physically associated biopolymer gels[49,50] and transient networks based on triblockcopolymers [51] have yielded higher values of D in therange 0.5–0.7. Interestingly, gels with semicrystallinedomains have lower exponents, including bacteriallyderived poly(b-hydroxyoctanoate) [52] and crystallizingpolypropylene [53]. Thus, the low value of D may be aconsequence of either entanglements of the PEO chainsor rigidity of the hydrophobic PLLA domains.

Fig. 6 shows the elastic modulus at fixed frequency asa function of strain for 2, 3, and 4 at 25�C. Thesepolymers exhibit a linear viscoelastic response over awide range of strain and applied stress, and no evidenceof strain-hardening was observed. Thus, we expect thesegels will have a uniform and predictable rheologicalresponse in vivo regardless of the mechanical environ-ment to which they are subjected.

4. Conclusions

We have reported, for the first time, a completerheological characterization of physically associatedhydrogels formed from PLLA–PEO–PLLA triblockcopolymers. The elastic modulus of these gels is stronglydependent on PLLA block length, offering a means ofcontrolling the mechanical properties for particular softtissue engineering applications. The triblock copolymersthat formed gels displayed elastic moduli in the range100–18,300 Pa at ambient and physiological tempera-tures, extending the upper limit of G0 that can beachieved with physical gels of PLA/PEO copolymers byan order of magnitude. The elastic moduli of our gelsare in the same range as several soft tissues, makingthese materials excellent candidates for a variety oftissue engineering applications.

Acknowledgements

This work was partially funded by and utilized centralfacilities of the NSF-sponsored UMass MRSEC onPolymeric Materials (DMR-0213695). G.N.T. thanksONR for a Young Investigator award, and S.R.B.thanks Dupont for a Dupont Young Professor Award.G.N.T. and S.R.B. both thank 3M for NontenuredFaculty awards.

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