ARTICLE IN PRESS

0142-9612/$ - se

doi:10.1016/j.bi

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Biomaterials 27 (2006) 5490–5501

www.elsevier.com/locate/biomaterials

Chemical and biological properties of supramolecular polymer systemsbased on oligocaprolactones

Patricia Y.W. Dankersa, Ellen N.M. van Leeuwena, Gaby M.L. van Gemertb,A.J.H. Spieringa, Martin C. Harmsenc, Linda A. Brouwerc, Henk M. Janssenb,

Anton W. Bosmand, Marja J.A. van Luync, E.W. Meijera,�

aLaboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, PO Box 513, NL-5600 MB Eindhoven, The NetherlandsbSyMoChem BV, Den Dolech 2, NL-5612 AZ Eindhoven, The Netherlands

cDepartment of Pathology and Laboratory Medicine, Medical Biology Section, University Medical Center Groningen, Hanzeplein 1,

NL-9713 GZ Groningen, The NetherlandsdSupraPolix Research Center, Horsten 1, NL-5612 AX Eindhoven, The Netherlands

Received 23 May 2006; accepted 13 July 2006

Abstract

We show that materials with a diverse range of mechanical and biological properties can be obtained using a modular approach by

simply mixing different ratios of oligocaprolactones that are either end-functionalized or chain-extended with quadruple hydrogen

bonding ureido-pyrimidinone (UPy) moieties. The use of two UPy-synthons allows for easy synthesis of UPy-modified polymers

resulting in high yields. Comparison of end-functionalized UPy-polymers with chain-extended UPy-polymers shows that these polymers

behave distinctively different regarding their material and biological properties. The end-modified UPy-polymer is rather stiff and brittle

due to its high crystallinity. Disks made of this material fractures after subcutaneous implantation. The material shows a low

inflammatory response which is accompanied by the formation of a fibrous capsule, reflecting the inertness of the material. The chain-

extended UPy-material on the contrary is practically free of crystalline domains and shows clear flexible properties. This material

deforms after in-vivo implantation, accompanied with cellular infiltration. By mixing both polymers, materials with intermediate

properties concerning their mechanical and biological behaviour can be obtained. Surprisingly, a 20:80 mixture of both polymers with

the chain-extended UPy-polymer in excess shows flexible properties without visible deformation upon implantation for 42 days. This

mixture, a blend formed by intimate mixing through UPy–UPy interaction, also shows a mild tissue response accompanied with the

formation of a thin capsule. The material does not become more crystalline upon implantation. Hence, this mixture might be an ideal

scaffold material for soft tissue engineering due to its flexibility and diminished fibrous tissue formation, and illustrates the strength of the

modular approach.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Foreign body response; Polycaprolactone; In vivo test; Scaffold; Self-assembly

1. Introduction

Important factors in biomaterial research are thetunability of the material properties with respect todegradability, bioactivity and mechanical behaviour. Forcertain applications in tissue engineering the right ingre-dients are needed to create the ideal scaffold material that

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.07.011

ing author. Tel.: +3140 2473101; fax: +31 40 2474706.

ess: e.w.meijer@tue.nl (E.W. Meijer).

fits the engineered tissue best [1,2]. A vast variety ofsynthetic [3] and natural polymers have been investigatedfor several medical applications such as tissue engineeringand drug delivery [4]. Polycaprolactone (PCL) is widelyused as biomaterial for transplantation purposes, but alsoas drug delivery vehicle in micro- and nanospheres [5]. PCLis known for its slow degradation profile; full degradationin vivo takes more than 2 years [6]. The degradation takesplace in a two-stage process. First, the ester groups arenon-enzymatically hydrolyzed and secondly, intracellular

ARTICLE IN PRESSP.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–5501 5491

degradation in macrophages and giant cells takes place [7].Many polymeric variations of PCL have been investigatedranging from blends [8] to co-polymers [9]. PCL was alsoused in shape-memory polymers, which can be used forminimally invasive surgery [10–12].

Recently, we have shown that a supramolecular andmodular toolbox can be used for the assembly of bioactivepolymers [13]. Using supramolecular building blocks, thatassemble non-covalently via specific interactions, makes itpossible to produce materials without tedious syntheticprocedures but simply by assembly. In this way, it becomeseasy to vary the amount and nature of the bioactivemolecules and the nature of the polymers. This supramo-lecular approach bridges the gap between covalentmodification and simple mixing of molecules and polymers.The first method leads to highly stable structures that lackdynamics. The latter, in contrast, results in very dynamicstructures that lack stability. Using a supramolecularapproach it is possible to control both stability anddynamics. The bioactive supramolecular system we haveshown before, is based on oligocaprolactone end-functio-nalized with 2-ureido-4[1H]-pyrimidinone (UPy) groups[13]. These UPy-moieties strongly dimerize via quadruplehydrogen bonding and display high association constants(Ka ¼ 106–107 Lmol�1) in organic solvents [14–17].

1 = == 2

(A)

(B)

(C)

(D)

Fig. 1. Schematic representations of the bifunctional and chain-extended UPy

the polymers, the second pictures are enlargements that indicate the dimerizatio

two dimerized UPy-units. (A) Oligocaprolactone (Mn ¼ 2.1 kg/mol) was end-

UPy-moieties at the chain-ends gives rise to the formation of virtual hig

(Mn ¼ 1.25 kg/mol) was chain-extended with UPy-units. This results in prepoly

These UPy-groups can dimerize which leads to the formation of a physical netw

by mixing UPy-polymers 1 and 2. This is proposed to result in UPy-dimers be

and PCL1250UPy6 2 that are used throughout this article.

The UPy-functionalized oligocaprolactone [13] showsmuch better mechanical properties than its unfunctiona-lized variation. These supramolecular polymer systems findalready many uses in polymer applications [18,19].Here, we report that the addition of more than two UPy-

units in the main chain can dramatically improve thematerial properties, while still supporting a modularapproach (Fig. 1). A chain-extended supramolecularoligocaprolactone-based UPy-polymer was designed(PCL1250UPy6 2; Fig. 1B) that can be easily blended withthe end-functionalized UPy-oligocaprolactone (PCL2000

UPy2 1; Fig. 1A). The material properties of the purepolymers were compared to mixtures of both polymers(Fig. 1C). Additionally, the in vivo behaviour was studiedshowing the tunability of the supramolecular system.

2. Materials and methods

2.1. General materials

The hydroxy-terminated PCLs (Mn ¼ 2.1kg/mol and Mn ¼ 1.25kg/mol)

were purchased from Acros. 1,6-Diisocyanatohexane was obtained from

Fluka. 2-Amino-4-hydroxy-6-methylpyrimidine, dibutyl tin dilaurate

(DBTDL) and isophorone diisocyanate (IPDI) were purchased from

Aldrich. Commercial products were used without further purification. All

solvents purchased from Acros Chimica or Sigma-Aldrich were of

6

N

N

O N

H

H

N

O

H

N

N

ON

H

HN

O

H

1

2

1+2

=

-modified polymers. (A–C) The first pictures show a schematic drawing of

n of the UPy-moieties and the third figure shows the chemical structure of

functionalized with UPy-moieties (PCL2000UPy2; 1). Dimerization of the

her molecular weight supramolecular polymers. (B) Oligocaprolactone

mers with approximately 6 UPy-units in the main-chain (PCL1250UPy6; 2).

ork. (C) A modular approach to supramolecular polymer mixtures is used

tween both prepolymers. (D) Schematic representations of PCL2000UPy2 1

ARTICLE IN PRESSP.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–55015492

p.a. quality. Deuterated chloroform was obtained from Cambridge

Isotope Laboratories. Water was always demineralized prior to use.

Phosphate-buffered saline (PBS) tablets were purchased from Sigma

(dissolution of the tablets in water resulted in a 0.01M phosphate buffer

with 0.0027M potassium chloride and 0.137M sodium chloride, with

pH ¼ 7.4).

2.2. General methods

1H-NMR and 13C-NMR spectra were recorded on a Varian Mercury

400MHz spectrometer at 298K. Chemical shifts are given in ppm (d)values relative to tetramethylsilane (TMS). Infrared (IR) spectra were

recorded on a Perkin Elmer Spectrum One FT-IR spectrometer with a

Universal ATR Sampling Accessory for solids. Elemental analysis was

carried out on a Perkin Elmer 2400 apparatus. Matrix assisted laser

desorption/ionization–time of flight (MALDI–TOF) mass spectroscopy

was performed on a PerSeptive Biosystems Voyager-DE PRO spectro-

meter using an acid a-cyano-4-hydroxycinnamic acid matrix. Gel

permeation chromatography (GPC) was performed on a Shimadzu

FCV-10 AL VP with a Shimadzu SCL-10AL VP system controller,

Shimadzu LC-10AD VP liquid chromatography pumps (with a PL gel

3mm mixed-E column; chloroform with 5% methanol as eluent or THF as

eluent), a Shimadzu DGU-14A degasser, a Shimadzu SPD-10AV UV-Vis

detector and a Polymer Laboratories PL-ELS 1000 detector. Narrow

polystyrene standards were used for calibration. Optical microscopy

pictures of the explanted disks were taken on a Zeiss Axiovert 25

microscope with a Sony (Cybershot, 3.3 Megapixels) digital still camera

DSC-S75 (Carl Zeis ACC Terminal). Normal photographs of these disks

were taken with the same Sony digital still camera DSC-S75. Pictures of

tissue slices were taken on a Leica DMLB microscope with Leica DC300

camera.

2.3. Synthesis UPy-hexyl-isocyanate synthon (1b)

The synthesis was performed as described before [15]. 2-Amino-4-

hydroxy-6-methylpyrimidine (1a; 0.23mol; 29.1 g) was dissolved in 1,6-

diisocyanatohexane (1.6mol; 272.3 g) and heated at 100 1C for 16 h.

Pentane (ratio 10:1 ¼ pentane:reaction mixture) was added to the reaction

mixture after cooling. The resulting precipitate was filtered and thoroughly

washed with pentane. The product was dried at 50 1C under reduced

pressure, yielding 1b as a white powder. Yield: 98%, 66.8 g, 0.23mol.

M.p.: 185 1C. 1H-NMR (CDCl3): d ¼ 13.14 (s, 1H, CH3–C–NH), 11.87 (s,

1H, CH2–NH–(CQO)–NH), 10.19 (t, 1H, CH2–NH–(CQO)–NH), 5.82

(s, 1H, CHQC–CH3), 3.27 (m, 4H, NH–(CQO)–NH–CH2+CH2–N-

CO), 2.23 (s, 3H, CH3), 1.61 (m, 4H, N–CH2–CH2), 1.41 (m, 4H,

CH2–CH2–CH2–CH2–CH2–CH2–NCO) ppm. 13C–NMR (CDCl3): d ¼173.1, 156.7, 154.8, 148.4, 106.8, 43.0, 39.9, 31.3, 29.4, 26.3, 26.2,

19.0 ppm. IR (ATR): n ¼ 2931, 2262 (NCO stretch), 1698 (UPy), 1667

(UPy), 1577 (UPy), 1519 (UPy), 1461, 1356, 1310, 1255 cm�1. Anal. calcd.

(%) for C13H19N5O3: C 53.2, H 6.5, N 23.9; Found (%): C 53.2, H 6.2,

N 24.0.

2.4. Synthesis PCL2000UPy2 (1)

PCL diol (Mn ¼ 2.1 kg/mol; obtained via ring-opening polymerization

initiated by diethylene glycol) was reacted with the UPy-hexyl-isocyanate-

synthon 1b, making use of a similar procedure as described before [15].

PCL diol (10mmol; 20.0 g) was dissolved in dry chloroform (300mL),

after which 1b (38mmol; 11.2 g) was added. After addition of two drops of

DBTDL the solution was stirred at 75 1C for 16 h. The completeness of the

reaction was checked with 1H-NMR and 13C-NMR for the presence of

OH endgroups. Then 5 g silica kieselgel 60 and two drops DBTDL were

added and the mixture was heated at 75 1C for 4 h. The reaction mixture

was put overnight at room temperature. The silica was removed by

filtration on a glasfilter over Hyflo after dilution of the mixture with

chloroform (1:1 ¼ chloroform:reaction mixture). With IR the absence of

1b in the solution was checked. Compound 1b was still present and the

solution was heated again with 3 g silica and two drops DBTDL at 75 1C

for 3 h. Again the silica was removed by filtration. The chloroform was

removed under reduced pressure; the material was precipitated from

chloroform in hexane and filtrated. The resulting white material was dried

for 2 days under reduced pressure. With IR the absence of 1b in the

material was checked again and it was found to be absent. Yield: 73%,

18.8 g, 7.3mmol. 1H-NMR (CDCl3): d ¼ 13.13 (s, 2H, C–NH–CQN,

UPy), 11.76 (s, 2H, CH2NH–(CQO)–NH, UPy), 10.14 (s, 2H,

CH2NH–(CQO)–NH, UPy), 5.85 (s, 2H, CQCH, UPy), 4.90 (s, 2H,

CH2NH–(CQO)–OCH2), 4.23 (t, 4H, CH2–(CQO)–OCH2CH2O), 4.06

(t, 2nH, CH2–(CQO)–OCH2), 3.69 (t, 4H, CH2–(CQO)–OCH2CH2O),

3.24 (m, 4H, CH2NH–(CQO)–NH), 3.16 (m, 4H, CH2NH–(CQO)–

OCH2), 2.31 (m, 2nH, CH2–(CQO)–OCH2), 2.23 (s, 6H, CH3, UPy), 1.64

(m, 4nH, CH2–(CQO)–OCH2CH2CH2CH2CH2), 1.50 (m, 16H, NH–

(CQO)–NH–CH2CH2CH2CH2CH2CH2–NH–(CQO)–O), 1.39 (m, 2nH,

CH2–(CQO)–OCH2CH2CH2CH2CH2) ppm. 13C–NMR (CDCl3): d ¼173.5, 173.1, 156.7, 156.5, 154.6, 148.2, 106.6, 69.0, 64.4, 64.1, 63.2,

40.6, 39.6, 34.0, 33.9, 29.7, 29.3, 28.7, 28.3, 26.2, 26.1, 25.5, 24.6, 24.5,

24.4, 18.9 ppm. IR (ATR): n ¼ 2943, 2865, 1721 (CQO stretch), 1698

(UPy), 1666 (UPy), 1584 (UPy), 1528 (UPy), 1471, 1419, 1397, 1366, 1294,

1240, 1185 (C–O stretch), 1107 cm�1.

2.5. Synthesis IPDI-UPy-IPDI synthon (2b)

5-(2-Hydroxyethyl)-6-methyl-isocytosine (2a) was synthesized from

2-acetylbutyrolactone according to the literature procedure [20]. 5-(2-

Hydroxyethyl)-6-methyl-isocytosine (2a; 71mmol; 12.0 g) was suspended

in IPDI (715mmol; 159 g) and was stirred for 16 h at 90 1C under an argon

atmosphere. A clear solution developed. The solution was cooled and

precipitated in hexane. The solid was filtered and stirred in another

portion of hexane. The product was isolated by filtration and washed with

hexane. The white residue was dried. Yield: 81%, 35.5 g; 58mmol. M.p.:

204 1C. 1H-NMR (CDCl3): d ¼ 12.93–12.83 (m, 1H, CH3–C–NH),

12.01–11.92 (m, 1H, CH2–NH–(CQO)–NH), 10.18–10.13 (m, 1H,

CH2–NH–(CQO)–NH), 5.15–4.54 (m, 1H, CH2–CH2–O–(CQO)–NH),

4.20 (t, 2H, CH2–CH2–O–(CQO)–NH), 3.98–3.26 (m, 3H, CH2–NCO

and CH–NCO), 3.10–2.92 (m, 3H, CH2–NH–(CQO)–O–CH2–CH2,

CH–NH–(CQO)–O–CH2–CH2, CH–NH–(CQO)–NH and CH2–NH–

(CQO)–NH), 2.79 (t, 2H, CH2–CH2–O–(CQO)–NH), 2.32 (s, 3H, CH3

at UPy), 1.89–1.74 and 1.30–0.90 (m, 4H; m, 26H, isophorone moieties).13C-NMR (CDCl3): d ¼ 171.7, 156.9, 155.5, 155.4, 153.1, 144.5, 122.4,

122.1, 121.5, 121.4, 121.3, 113.5, 62.4, 56.8, 56.7, 56.5, 52.9, 50.4, 48.6,

48.3, 48.2, 47.9, 47.7, 47.5, 46.3, 46.2, 45.9, 45.8, 45.7, 45.5, 45.0, 44.2,

43.5, 43.4, 43.0, 42.1, 41.7, 41.2, 40.2, 36.7, 36.4, 36.3, 36.2, 36.1, 34.6,

34.5, 34.3, 31.6, 31.5, 31.4, 31.3, 31.2, 29.8, 28.9, 27.3, 27.2, 26.6, 26.5,

25.3, 23.1, 23.0, 22.3, 16.9, 13.8 ppm. IR (ATR): n ¼ 2955, 2259 (NCO

stretch), 1694 (UPy), 1661 (UPy), 1588 (UPy), 1532 (UPy), 1463, 1364,

1306, 1252 cm�1. MALDI–TOF MS: m/z: Calcd. 613.8 g/mol. Obsd.

[M+H]+ ¼ 614 g/mol, [M+Na]+ ¼ 636 g/mol. For convenience, only

one isomer of the product is shown. IPDI exists in different regio- and

stereoisomers, and the coupling is not selective for one of the isocyanates

in IPDI.

2.6. Synthesis PCL1250UPy6 (2)

Telechelic hydroxy-terminated PCL (Mn ¼ 1.25 kg/mol; obtained via

ring-opening polymerization initiated by diethylene glycol) was dried in

vacuo. The PCL diol (21 mmol; 25.9 g) was reacted with the IPDI–

UPy–IPDI synthon 2b (18mmol; 10.9 g) in dry ethylacetate (130mL) in

the presence of two drops of DBTDL for 16 h at 70 1C. After that,

ethylacetate and ethanol (70mL:50mL) were added to the reaction

mixture, which was subsequently precipitated twice into ethanol. The

polymer was isolated after drying, resulting in a white elastic material.

Yield: 90%, 33.1 g, 2.7mmol. 1H-NMR (CDCl3): d ¼ 12.84 (s, 1H,

CH3–C–NH), 11.88 (s, 1H, CH2–NH–(CQO)–NH), 10.03 (s, 1H,

CH2–NH–(CQO)–NH), 4.95–4.40 (3s, broad, 2H, NH–(CQO)–O),

ARTICLE IN PRESSP.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–5501 5493

4.16 (t, 4H, CH2–CH2–O–(CQO)–NH and CH2–(CQO)–OCH2CH2O),

3.99 (t, 2nH, CH2–(CQO)–OCH2), 3.71 (t, 2H, CH2–(CQO)–OCH2-

CH2O), 3.61 (t, 4H, CH2–NH–(CQO)–O–(CH2)5) and CH–NH–

(CQO)–O–(CH2)5), 3.19–2.66 (t, 2H, CH2–CH2–O–(CQO)–NH; m,

6H, CH2–NH–(CQO)–O–CH2–CH2, CH–NH–(CQO)–O–CH2–CH2,

CH–NH–(CQO)–NH, CH2–NH–(CQO)–NH), 2.24 (m, 2nH, CH2–

(CQO)–OCH2; s, 3H, CH3 at UPy), 1.56 (m, 4nH, CH2–(CQO)–OCH2CH2CH2CH2CH2; m, 4H, isophorone-moieties), 1.30 (m, 2nH,

CH2–(CQO)–OCH2CH2CH2CH2CH2), 1.12–0.86 (m, 26H, isophorone

moieties) ppm. Note (1H-NMR): the integrals are based on 1 UPy-group

and 1 prepolymer PCL-chain. 13C-NMR (CDCl3): d ¼ 173.5, 173.4, 156.9,

155.2, 69.1, 64.1, 63.3, 46.8, 44.5, 36.4, 35.1, 34.1, 33.9, 32.3, 31.8, 28.7,

28.3, 27.6, 25.5, 25.4, 24.6, 24.5, 24.4, 23.2 ppm. IR (ATR): n ¼ 2945,

2866, 1722 (CQO stretch), 1700 (UPy), 1661 (UPy), 1586 (UPy), 1530

(UPy), 1460, 1419, 1390, 1366, 1295, 1240, 1190 and 1162 (C–O stretch),

1105 cm�1.

2.7. Preparation of mixtures

Films were made of the pristine polymers PCL2000UPy2 1 and

PCL1250UPy6 2 and of mixtures consisting of different ratios (in weight%)

by dissolution of the polymers separately or simultaneously (in the case of

mixtures) in THF while stirring the solution for 16 h at room temperature

(concentration ¼ 0.1 g/mL). The polymer solutions were drop cast in

teflon moulds. The generated films were dried in vacuo for 1–2 days at

37 1C. Mixtures consisting of 60:40 (m1), 50:50 (m2) and 20:80 (m3)

PCL2000UPy2: PCL1250UPy6 were used. Mixtures m1 and m3 were used to

perform the tensile testing experiments and in vivo studies. The materials

that were used for in vivo experiments were sterilized with UV for at least

2 h on each side.

2.8. Tensile testing

Mechanical properties were performed in a tensile mode according to

ASTM D1708-96 specifications in air at room temperature on a Zwick

Z010 Universal Tensile Tester at an elongation rate of 20mm/min with a

preload of 0.02 N and a load cell of 20 N. Tensile bars with a cross section

of approximately 1.5mm2 were obtained by punching them out of sheets

of materials that were prepared by drop casting out of THF. The grip-to-

grip separation was approximately 20mm. The measurements were

performed at least in quadruplicate. Yield stresses and yield strains were

determined, due to the shape of the stress–strain curves, by taking the

intersection point of the two tangents to the initial and final parts of the

load elongation curves around the yield point [21]. An indicative Young’s

modulus (E) was determined by calculating the slope at 0% strain.

2.9. Differential scanning calorimetry

Thermal properties were investigated with differential scanning

calorimetry (DSC) on a Perkin Elmer Differential Scanning Calorimeter

Pyris 1 with Pyris 1 DSC Autosampler and Perkin Elmer CCA7 cooling

element under a nitrogen atmosphere with heating and cooling rates of

20 1C/min (samples of 10–12mg were measured). The temperature range

was �100 1C to120 1C.

2.10. In vivo implantations: PCL2000UPy2 versus PCL1250UPy6

Solution cast films of PCL2000UPy2 1 and PCL1250UPy6 2 (diame-

ter ¼ 6mm, thickness ¼ approx. 0.4mm) were subcutaneously implanted

into male Albino Oxford (AO) rats, after sterilization of the disks with UV

for at least 2 h on each side. The rats had an age of approximately 10–12

weeks. Animal experiments were carried out according to the NIH

guidelines for care and use of laboratory animals. The rats were

anesthetized with a mixture of halothane, N2O and O2. The animals were

shaved and disinfected. The subcutaneous pockets were made on the back

of the rat, three on the right and three on the left side. One sterilized disk

was placed in each pocket. The animals were housed in a temperature-

controlled and humidity-controlled room with 12 h light/dark cycles after

the surgery. They had access to water and standard rat food. The rats were

anesthesized before explantation of the disks after certain time points. The

rats were sacrificed. In total 10 rats were used. The implants with the

surrounding tissue were explanted after 2, 5, 10, 21 and 42 days of

implantation and were fixed in 2% glutaraldehyde solution and embedded

in plastic (Technovit 7100 cold curing resin based on hydroxyethylmetha-

crylate (HEMA), Kulzer Histo-Technik). The tissue slices (2 mm) were

stained with toluidine blue for histological examination with optical

microscopy.

2.11. In vivo implantations: mixtures

Solution cast films of polymers 1 and 2 and of mixtures m1 and m3 of

both polymers in different ratios (60:40 and 20:80 PCL2000

UPy2:PCL1250UPy6, for m1 and m3, respectively) were subcutaneously

implanted for 42 days as described above. In total 7 rats were used. The

materials and surrounding tissues were processed in three ways in

triplicate for the different samples. The surrounding tissues of the first

fraction of materials were removed after which the disks were rinsed with

water and dried for 1.5 h at 30 1C. After that, different techniques were

used to study these samples; mass loss (the dry mass was measured on a

Sartorius microbalance), morphology, NMR (conc. 14mg/mL in CDCl3),

IR and DSC measurements were performed. The second fraction was

embedded in plastic (see above) and stained with toluidine blue. The third

part was frozen in liquid nitrogen and cut into slices (7mm) for immune

staining with anti-collagen IV antibody (goat anti-type IV collagen-

UNLB, Southern Biotechnology Associates), the ED1 marker (mouse

anti-rat CD68 MCA341 R, Serotec) and the R73 antibody. The second

antibodies that were used, were RAMPO (polyclonal rabbit anti-mouse

immunoglobulins HRP, DakoCytomation) for ED1 and R73 and

RAGPO (polyclonal rabbit anti-goat immunoglobulins HRP, DakoCy-

tomation) for collagen IV. The final colouring reaction was performed

with hydrogen peroxide and 3-amino-9-ethylcarbazole (AEC) resulting in

a red colour if the staining was positive. Finally, the tissue slices were

coloured with hematoxilin (blue). As positive control tissue rat spleen was

used.

2.12. In vitro incubation

The pristine polymers 1 and 2 and the mixtures m2 and m3 (90–140mg

per sample) were incubated in PBS buffer (20mL per sample) in duplicate

for 126 days at 37 1C. Mass measurements (the wet mass (water

absorption) and dry mass (mass loss) of the samples were measured on

a Sartorius microbalance) and DSC experiments were performed, after

rinsing the samples three times with water and drying them at 30 1C for

1.5 h.

3. Results and discussion

3.1. Synthesis of UPy-synthons and UPy-polymers

Both materials, the bifunctional and chain-extendedUPy-polymers, were synthesized using two different UPy-isocyanate synthons, to facilitate the synthetic method.The bifunctional UPy-terminated PCL (PCL2000UPy2 1;Scheme 1A) consists of a PCL part with a molecular weightof 2.1 kg/mol. Methyl-isocytosine (1a) was reacted for 16 hat 100 1C in an excess of hexyldiisocyanate resulting in theformation of the UPy-hexyl-isocyanate synthon, 2(6-isocyanatohexylamino)-6-methyl-4[1H]-pyrimidinone (1b)[15]. The UPy-hexyl-isocyanate synthon 1b was precipi-tated in pentane and was obtained as a white powder with

ARTICLE IN PRESS

OHO

OO

OH

O O

n n

OCNNCO

N

NH

NH

NH

O

O

NCO

N

NH

NH

NH

O

O

NH

OO

O

O

On

N

NH

NH2O

N

NHOHO

OO

O NH

O O

O

NH

O

OO N

HNH

O

NH

O

O

OO

O

O O

OH

n n

n n

N

NH

O NH

O

NH

O

NCO

NH

O

OCNOCN

NCO

NH

N

O NH2

OH

OHO

OO

OH

O On n

100°C

90 °C EtOAc, 70°C, DBTDL

CHCl3, 75°C, DBTDL

1

(A)

(B)

6

2

1a 1b

2b2a

2

6

Scheme 1. Synthesis of the bifunctional and chain-extended polymers. (A) Synthesis of PCL2000UPy2 (1); g ¼ 98% (1b) and g ¼ 73% (1). (B) Synthesis of

PCL1250UPy6 (2); g ¼ 81% (2b) and g ¼ 90% (2). The IPDI unit exists in different regio-isomers and stereo-isomers and the coupling is not selective for

one of the isocyanates of IPDI. Therefore, for convenience, only one possibility of the IPDI–UPy–IPDI synthon (2b) is shown.

1

2m1

m3

Fig. 2. Tensile test data of the different polymer films that were used for

subcutaneous implantation in rats. The films were made of 100%

PCL2000UPy2 (1) and 100% PCL1250UPy6 (2) or of mixtures with the

following ratios 60:40 (m1) and 20:80 (m3) PCL2000UPy2:PCL1250UPy6.

P.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–55015494

a yield of 98%. Hydroxy-terminated PCL was reacted withan excess of 1b in chloroform with DBTDL as catalyst at75 1C for 16 h resulting in PCL2000UPy2 (1) [13,15]. Themixture became more viscous in the course of the reaction,indicating the formation of virtual high molecular weightpolymers due to dimerization of the UPy-moieties (Fig.1A). The excess of 1b was removed via stirring the reactionmixture with silica at 75 1C in the presence of DBTDL for4 h. The silica was removed by filtration and 1 wasprecipitated in hexane [13,15] and obtained as a whitematerial with a yield of 73%.

The molecular weight of the PCL prepolymer used forthe chain-extended PCL (PCL1250UPy62; Scheme 1B) is1250 g/mol. On average, six UPy-units are part of themain chain. The starting compound 5-(2-hydroxyethyl)-6-methyl-isocytosine (2a) was synthesized from 2-acetylbu-tyrolactone [20]. Compound 2a was reacted in an excessof IPDI for 16 h at 90 1C to yield IPDI–UPy–IPDIsynthon 2b. The product was precipitated in hexane, whichresulted in 81% 2b as a white powder. The hydroxyend-groups of the PCL (1.25 kg/mol) were reacted with2b in a ratio of 7:6 in ethylacetate in the presence ofDBTDL as catalyst for 16 h at 70 1C. Also, during thisreaction the mixture became more viscous, indicating theformation of longer polymers via chain extension and thegeneration of physical aggregation between chains byUPy–UPy dimers (Fig. 1B). Polymer 2 was precipitatedin ethanol and obtained as a white elastic material with ayield of 90%.

3.2. Mechanical properties

Mixtures consisting of different ratios (in weight%) ofpolymer 1 and 2 (60:40 (m1), 50:50 (m2) and 20:80 (m3)PCL2000UPy2:PCL1250UPy6) were used to correlate differ-ent material properties to their in vitro and in vivobehaviour. Tensile test experiments were performed on

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Table 1

Tensile test data of the polymer films that were used for subcutaneous implantation in rats. The Young’s modulus (E in MPa), yield stress (syield in MPa),

yield strain (eyield in %), stress at break (sbreak in MPa), elongation at break (ebreak in %) and the maximum stress (smax in MPa) are shown

E (MPa) syield (MPa) eyield (%) sbreak (MPa) ebreak (%) smax (MPa)

1 12977 6.370.25 5.470.1 6.070.4 1474 6.570.4

m1 28710 2.270.5 9.170.9 2.070.2 33718 2.470.7

m3 4.170.7 0.7270.08 35.276.5 0.970.1 5117143 1.070.1

2 3.170.6 0.8370.17 45.874.6 2.470.6 5767172 2.570.6

P.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–5501 5495

the pristine polymers (1 and 2) and on the differentmixtures (60:40 and 20:80 PCL2000UPy2:PCL1250UPy6,m1 and m3, respectively) to investigate their mechanicalproperties.

The differences in mechanical properties between poly-mer 1 and 2 are clear (Fig. 2; Table 1). In the case ofpolymer 1, the Young’s modulus (E) and yield stress (syield)are relatively high, whereas the elongation at break (ebreak)is relatively low. Polymer 1 is rather stiff, more brittle andless tough as compared to polymer 2, which is more flexibleand ductile. Polymer 1 is able to form relatively high virtualmolecular weight chains via hydrogen bonding between theend-capped UPy-groups (Fig. 1A). This dimerization givesthe material its properties; the unfunctionalized hydroxy-terminated oligocaprolactone is a waxy, brittle solid. Onthe contrary, polymer 2 forms physical cross-links betweenthe already longer chains that are formed by covalentchain-extension during synthesis resulting in a hugephysical network (Fig. 1B). Mixtures of polymer 1 and 2

are visualized in Fig. 1C.The tensile tests on the mixtures show that after addition

of 40% 2 to 1 (m1), the E and syield decrease, that ebreakincreases and that the yield point (eyield) takes place at alarger elongation. This indicates that material m1 is lessstiff, and more tough and flexible than 1. Noteworthy isalso that the graph of m1 slowly moves in time to theposition of 1, which might be caused by aging andcrystallization. This is also reflected in the relatively highermargins of error for m1 (Table 1). Surprisingly, when more2 is added to 1, a material (m3) is obtained that has a loweryield stress (syield) and stress at break (sbreak) whencompared to 2. Evidently, the relative low amount of 1 isnot enough to increase the syield and sbreak of 2, and theopposite is observed. This might be caused by the fact thatthe presence of 1 hampers the strain-induced crystallizationthat is present to some extent in pristine 2. This makes thematerial m3 weaker (by reduction of the stress at break).It is evident that the material properties can be tuned bymixing 1 and 2.

3.3. Thermal properties

The thermal properties of the polymers 1 and 2 and ofthe mixtures m1 and m3 were studied with DSC. Cleardifferences are visible between the second heating runs ofthe different polymers and polymer mixtures, reflecting the

intrinsic polymer properties (Fig. 3A and B; supplementaryinformation). The unfunctionalized PCL prepolymers donot show a glass transition temperature (Tg; data notshown). However, owing to UPy-functionalization a Tg

appears for all polymers studied. The Tg shifts to highertemperatures upon increasing the relative amount of 2 inthe mixtures (Fig. 3B). This reflects the intimate mixingbetween 1 and 2, showing that the polymers do not phaseseparate. The shifting of the Tg is possibly caused by thepresence of a larger amount of longer chains, which leadsto a decrease of the free volume. A decrease in the heatcapacity, DCp, was measured upon addition of 2 (Fig. 3B).Only the films consisting of 1 and m1 showed a meltingpeak (Tm) in the second heating run around 42 1C with amelting enthalpy, DHm of 31 and 1.1 J/g, respectively. Thislowering of DHm for m1 might be caused by suppression ofthe crystallization of 1 by 2. This might be due to theshorter PCL chains of 2. Another possibility is that theIPDI–UPy–IPDI units in 2 are less prone to crystallizationthan the UPy-hexyl moieties in 1, because of stericreasons and/or the presence of different ways of IPDIincorporation.The films of 2 and m3 did not show a melting peak. The

tendency of the PCL part to crystallize is higher for thePCL2000UPy2 polymer 1 than for the PCL1250UPy6polymer 2, which is mainly caused by the chain length ofthe PCL part. The absence of a Tm for 2 and m3 might alsobe caused by the presence of the IPDI–UPy–IPDI moieties.The thermogram of 1 showed a second melting peak at58 1C. This peak is probably originating from the meltingof the UPy-hexyl-urethane dimer hard-blocks (the UPy–UPy stacks in the lateral direction). This peak might alsobe due to a second, more thermodynamically stable,crystalline form of PCL. This peak was not found formixture m1, which is probably because of the presence ofthe long PCL1250UPy6 chains that create less orderedstructures or caused by the IPDI–UPy–IPDI moieties.Only polymer 1 shows a crystallization (Tc) signal in thesecond heating run.The DSC results discussed here show that as expected

polymer 1 and 2 do not phase separate, which is possiblyenhanced by the UPy–UPy interaction. However, phaseseparation between the UPy-units and PCL-chains mightbe present. To search for a material with both excellentmaterial properties and good biological properties, thesemixtures were studied in vivo.

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ratRT

2nd heating run(A) (B)

(C) (D)

2nd heating run

1st heating run 1st heating run

amount 2 (%)

amount 2 (%)

2

2

1

1

m1

m1

m3

m3

22

11

m1

m1

m3

m3

ratRT

ratRT

temperature (°C)

temperature (°C)

delta

Cp

(J/g

°C)

del

ta H

m (

J/g

°C)

Tg

(°C

)

hea

t fl

ow

(A

.U.)

h

eat

flo

w (

A.U

.)

Fig. 3. Thermal properties of the pristine polymers PCL2000UPy2 (1) and PCL1250UPy6 (2) and of mixtures consisting of 60:40 (m1) or 20:80 (m3)

PCL2000UPy2:PCL1250UPy6 after 42 days at room temperature (RT) and after 42 days of subcutaneous implantation in rats (rat). DSC was measured at

20 1C/min. (A) Thermogram of the second heating run, indicating the intrinsic polymer properties. (B) The glass transition temperatures (Tg) and heat

capacities (DCp) as function of the amount of 2 mixed with 1. These values were taken from the second heating run of the polymers that were left for 42

days at room temperature. (C) Thermogram of the first heating run, showing the thermal history of the materials. (D) The melting enthalpies (DHm) as

function of the amount of 2 mixed with 1. These values were taken from the first heating run.

P.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–55015496

3.4. Tissue response of end-modified and chain-extended

UPy-polymers

The pristine polymers 1 and 2 were subcutaneouslyimplanted as disks in rats to study their biocompatibilityand the onset, progression and resolution of the ForeignBody Reaction (FBR) in vivo. Despite the fact that theFBR is very low for both materials, differences between thetwo polymers were clearly visible on the level of cellularinfiltration and immune response (Fig. 4). PCL2000UPy2 1seemed chemically inert and stayed intact during the wholeperiod. Fibrin was observed at the interface up to 5 daysfor both polymers (f; Fig. 4A—1 and A—2). Remarkably,after 10 days PCL1250UPy6 2 seemed to be slightlydeformed. This deformation became more intense after21 and 42 days. This phenomenon was attended byincreased cellular infiltration of macrophages (Mfin;Fig. 4B—2) and giant cells (Gcin; Fig. 4B—2) in time. Itis not inconceivable that this distortion of the shape of

material 2 might be partially due to the ingrowth of thesecells (supplementary information). This infiltration isprobably caused by its low crystallinity (see below). Incontrast, for 1, macrophage infiltration accompaniedby some exudate formation was observed at day 2. Thepresence of macrophages at the interface became relativelylow after 21 days (Mfin; Fig. 4B—1). Giant cells weresporadically present at the interface (Gcin; Fig. 4B—1). Aclear fibrous capsule (c; Fig. 4A—1) was developed for 1

from day 10 up to 42, with an increased amount offibroblasts (F; Fig. 4B—1). On the contrary, for 2 thecapsule (c; Fig. 4A—2) was rather thin and the amountof fibroblasts (F; Fig. 4B—2) was low. Also, anotherdistinction could be made concerning the vascularization inthe surrounding tissue (Vas; Fig. 4B—1 and 4B—2). Ingeneral, vascularization was low for 2, while increased for 1after 10 days. However, vascularization seems to beincreased at the interface of 2. For both polymers 1 and2 holds that polymorph nuclear cells and lymphocytes were

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Fig. 4. In vivo behaviour of the pristine polymers PCL2000UPy2 (1) and PCL1250UPy6 (2) after different time points (2, 5, 10, 21 and 42 days) of

subcutaneous implantation in rats. (A) Histological examination after toluidine blue staining. The following abbreviations are used: m ¼ material,

v ¼ blood vessel, f ¼ fibrin, c ¼ fibrous capsule, # ¼ macrophage, * ¼ giant cell. All scale bars represent 100mm. (B) The amount of different cell types

and vascularization were determined in the corresponding tissue sections. Scores (in A.U.) between 0 and 10 were given; 0 means not present and 10

signifies abundantly available in the tissue section. The following abbreviations are used: Gcin ¼ giant cells at the interface, Mfin ¼ macrophages at the

interface, Mfout ¼ macrophages in the capsule and in the surrounding tissue, F ¼ fibroblasts in the fibrous capsule and in the surrounding tissue,

Vas ¼ blood vessels/vascularization in the capsule and in the surroundings.

P.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–5501 5497

scarcely present in the surrounding tissue at all time points,which again shows that the FBR is very mild for bothmaterials.

The mild FBR in the case of material 1 is accompaniedwith fibrous capsule formation showing the inertness of thematerial. On the contrary, for material 2 major cellularinfiltration, accompanied by a very thin fibrous capsule and

an increased amount of vascularization at the interface, isobserved. It was already demonstrated for other polymerscaffolds that an increased cellular invasion can beaccompanied by higher vascularization [22]. Besides thislow FBR, major deformation could be detected for 2. Sothis material might find its application as self-adaptingwound filler.

ARTICLE IN PRESSP.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–55015498

3.5. Mass loss of implanted mixtures

A modular approach was applied to tune deformationand cellular infiltration. Consequently, both pristinepolymers 1 and 2 and mixtures with different ratios,60:40 (m1) and 20:80 (m3) PCL2000UPy2:PCL1250UPy6were implanted subcutaneously for 42 days to study theresolution of the FBR. As a control experiment, thepristine polymers 1 and 2 and mixtures 50:50 (m2) and20:80 (m3) were incubated in vitro in PBS buffer at 37 1Cfor 126 days.

The degradation of the implanted disks was investigatedby measuring mass loss. The implanted disks were notdegraded during the period of 42 days according to massloss experiments (0.870.2% for 1, 1.170.3% for m1,0.670.1% for m3 and 0.0970.04% for 2). This was alsoconfirmed with 1H-NMR. The spectra were exactly thesame before and after implantation for 42 days; nostructural differences or hydrolysis could be detected.Also, GPC did not show any hydrolysis. This in vivoexperiment was compared to films made of the purepolymers 1 and 2 and of similar mixtures of PCL2000

UPy2:PCL1250UPy6 (50:50 (m2) and 20:80 (m3)) that wereincubated for 126 days in PBS buffer (pH 7.4) at 37 1C.Upon addition of 2 the water absorption might becomehigher when compared to the pure 1 after 126 days (373%for 1, 772% for m2, 471% for m3 and 673% for 2).Unfortunately, the error in these measurements is largewhich prohibits real quantification. Furthermore, the massloss in vitro is lower for mixtures with more 2 as comparedto the pure 1 (574% for 1, 171% for m2, 0.270.2% form3 and 0.370.1% for 2). This might be caused by the factthat 1 becomes more crystalline during incubation than 2

(see below) which leads to partial fragmentation of the filmcaused by increased brittleness of 1. However, even after126 days in buffer the mass loss is negligible small for allfilms. In conclusion, the end-modified and chain-extendedUPy–PCL polymers do not degrade during 42 daysof subcutaneous implantation, nor in buffer at 37 1C for126 days.

3.6. Morphology and crystallinity of implanted mixtures

The morphology and crystallinity of the mixtures afterimplantation was studied using optical microscopy, IRspectroscopy and DSC. The morphology of the diskschanges during implantation (Fig. 5A). The disks of 1 andm1 are intact but break upon explantation, reflecting thebrittle nature of the materials due to their higher crystal-linity. Both materials can be removed easily from thecapsule, indicating hardly any interaction with the cells.The surface of these materials is very smooth, which is alsovisible using optical microscopy (Fig. 5A). Material m3

stays intact and is not deformed. Disk 2 is much moredifficult to remove from its capsule and shows on thecontrary a massive deformation (Fig. 5A). The less smoothsurface of 2 is confirmed with optical microscopy (Fig. 5A).

To study the surface morphology, the IR spectra ofpolymers 1 and 2 and mixtures m1 and m3 that were keptfor 42 days at room temperature were compared to thespectra of the samples that were subcutaneously implantedin rats for 42 days. The IR spectra for all polymer filmswere similar (supplementary information). The spectra ingeneral became broader owing to implantation, especiallyfor disks 2, m1 and m3, indicating that the system showsless organization at the surface [23]. This more randomstate can be attributed to the presence of water, which isshown at 3300 cm�1. This characteristic OH band becamehigher after implantation. Water might be incorporated ordisturb the hydrogen bonding between the UPy-groups.However, no evidence in this study or earlier investigationsis found for a significant effect of water on the quadruplehydrogen bonding units. The IR measurements also showthat the disks do not degrade during the investigated time.To investigate the nature of the fracturing and deforma-

tion of the disks after implantation, the thermal propertiesof the implanted disks were measured. From the secondheating runs (Fig. 3A) it is clear that the polymers 1 and 2

and mixtures m1 and m3 display similar phase behaviourafter subcutaneous implantation for 42 days in rats whencompared to the samples that were kept at roomtemperature for 42 days. Very similar results were foundfor the samples (1, 2, m2 and m3) that were incubated inbuffer for 126 days at 37 1C (supplementary information).Whereas the intrinsic polymer properties do not change,clear differences are found concerning the thermal historyof the disks retrieved from the first heating runs (Fig. 3Cand D). The glass transition temperatures (Tg) stay more orless the same and follow the same trend as was observed forthe second heating runs. Unfortunately, the heat capacitiescannot always be determined. The onset of the meltingtemperature changes after incubation from approximately30 1C to temperatures around 50 1C. This implies theformation of more stable PCL crystals upon annealing at37–38 1C in the rat in all four films. The same results werefound for the polymers 1, 2, m2 and m3 that wereincubated in vitro (supplementary information) indicatingthat these phenomena are not due to subcutaneousimplantation but probably caused by annealing at 37 1Cand the presence of an aqueous environment.The materials 1, 2 and m1 become more crystalline upon

incubation; an increase in DHm can be seen. The increase inDHm for 1 is much larger than for the other mixtures.Striking is that the melting enthalpies, DHm for thepolymers that were kept at room temperature, m1 andm3 are higher than for 1, indicating that the crystallizationprocess at room temperature is slower for 1. Afterannealing in rats or in buffer these differences change.Hardly any change in DHm for mixture m3 is found afterannealing in rats or in buffer as compared with the samplesthat were left at room temperature (Fig. 3D). This indicatesthat m3 is already in its most thermodynamically stablestate before annealing. So, disks made of 1 and m1 becamemore crystalline owing to subcutaneous implantation

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Fig. 5. In vivo behaviour of the disks of the pristine polymers PCL2000UPy2 (1) and PCL1250UPy6 (2) and of mixtures consisting of 60:40 (m1) or 20:80

(m3) PCL2000UPy2:PCL1250UPy6 after 42 days of subcutaneous implantation in rats. (A) The explanted disks. The first row consists of photographs. The

second row shows optical micrographs with enlargements at 100 times magnification. All scale bars represent 1mm. (B) Histological examination after

toluidine blue staining; m indicates the material. The scale bars represent 100mm. (C) Histological examination after toluidine blue staining. The following

abbreviations are used: m ¼ material, v ¼ blood vessel, c ¼ fibrous capsule, # ¼ macrophage, * ¼ giant cell. The scale bars represent 100mm. (D)

Immune-stained histology. The first row shows tissue sections that were stained using the anti-collagen IV antibody to detect vascularization. The second

row consists of micrographs of tissue sections stained with the ED1 marker against macrophages. The third row shows tissue sections were stained with the

R73 antibody against T-cell receptors on the lymphocytes. The scale bars represent 100mm. m ¼ material, v ¼ blood vessel, c ¼ fibrous capsule,

# ¼ macrophage, * ¼ giant cell.

P.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–5501 5499

ARTICLE IN PRESSP.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–55015500

(displaying similar DHm; Fig. 3D) reflecting their brittle-ness and fracture upon explantation. The change incrystallinity of disk 2, however, was relatively low. Thisphenomenon was accompanied by the very small DHm,which resulted in deformation of disk 2. On the contrary,no change in crystallinity could be detected for m3, but itsDHm was high enough to resist deformation.

3.7. Tissue response of mixtures

The histology of the tissue sections of the mixtures wasinvestigated to determine the cellular response. Thetoluidine blue-stained tissue sections reveal that 1 is aninert material and no cellular infiltration into the materialwas observed, as already shown above (Fig. 5B and C).This is in accordance with the ease of explantation. Forpolymer 2 the cellular infiltration is clearly visible byshowing macrophages and giant cells at the interface(Fig. 5C and D). The amount of different cell types andvascularization were determined in the correspondingtissue sections of the mixtures m1 and m3 by immuno-histochemistry and were compared to the pristine polymers1 and 2 (Figs. 5D and 6).

Mixture m3 is more comparable to polymer 2. In the caseof polymer 2 the amount of macrophages (Mfin; Figs. 5Dand 6) at the interface is much higher and also giant cells(Gcin; Figs. 5D and 6) are visible. This surface reaction isalso present for m3 but to a lesser extent. Mixture m1

shows a similar FBR as polymer 1 (Fig. 6). The fibrouscapsule comprising of many fibroblasts is thicker for 1, m1

and m3 (F; Fig. 6). Hardly any macrophages are present inthese capsules (Mfout; Figs. 5D and 6). Also the amount of

F VasGcin

Fig. 6. The amount of different cell types and vascularization were

determined in the tissue sections of the pristine polymers PCL2000UPy2 (1)

and PCL1250UPy6 (2) and of mixtures consisting of 60:40 (m1) or 20:80

(m3) PCL2000UPy2:PCL1250UPy6 after 42 days of subcutaneous implanta-

tion in rats. Scores (in A.U.) between 0 and 10 were given; 0 means not

present and 10 signifies abundantly available in the tissue section. The

following abbreviations are used: Gcin ¼ giant cells at the interface,

Mfin ¼ macrophages at the interface, Mfout ¼ macrophages in the

capsule and in the surrounding tissue, F ¼ fibroblasts in the fibrous

capsule and in the surrounding tissue, Vas ¼ blood vessels/vascularization

in the capsule and in the surroundings.

macrophages located in the surrounding tissue is very low(Mfout; Figs. 5D and 6). Furthermore, lymphocytes arenot present in all material explants (Fig. 5D). Thevascularization in the surroundings is much higher forpolymer 1 and m1 (Vas; Figs. 5D and 6). For material 2,vascularization is particularly higher at the interface. In thecase of disks 1, m1 and m3 a more granular anti-collagenIV staining near the interface is observed (Fig. 5D). Thismight indicate desintegration of the extracellular matrix ofblood vessels. It is clear that the inflammatory response onthe different polymeric disks can be tuned via mixingpolymer 1 and 2. Upon addition of more 2 in the mixturethe response becomes more located to the surface. Thisreaction is accompanied with higher vascularization nearthe interface and a thinner fibrous capsule.

4. Conclusions

Intimate mixing through quadruple hydrogen bondingallows the formation of systems based on two oligocapro-lactone-based building blocks: end-functionalized UPy-oligocaprolactone (PCL2000UPy2) and chain-extendedUPy-oligocaprolactone (PCL1250UPy6). The two polymerbuilding blocks show different material properties andbiological behaviour in vivo. Making use of all the benefitsof these reversible supramolecular polymer systems, ahighly interesting biomaterial was formed, comprising of20:80 PCL2000UPy2:PCL1250UPy6. This mixture not onlydisplays good mechanical properties as it does not deformor become more crystalline after subcutaneous implanta-tion, but also the foreign body reaction displayed is verymild.In this way it is shown that with a modular approach the

material properties and biological in vivo behaviour can betuned. This approach opens the way to develop scaffoldswith material properties tuned to the specific needs of theapplication and to use the UPy–UPy interactions toincorporate UPy-modified bioactive factors without theneed for covalent modification.

Acknowledgement

This work is supported by the Council for ChemicalSciences of the Netherlands Organization for ScientificResearch (CW-NWO).

References

[1] Griffith LG, Naughton G. Bodybuilding: the bionic human: tissue

engineering—current challenges and expanding opportunities.

Science 2002;295:1009–14.

[2] Hench LL, Polak JM. Bodybuilding: the bionic human: third-

generation biomedical materials. Science 2002;295:1014–7.

[3] Agrawal CM, Ray RB. Biodegradable polymeric scaffolds for

musculoskeletal tissue engineering. J Biomed Mater Res 2001;55:

141–50.

[4] Langer R, Peppas NA. Advances in biomaterials, drug delivery, and

bionanotechnology. AIChE J 2003;49:2990–3006.

ARTICLE IN PRESSP.Y.W. Dankers et al. / Biomaterials 27 (2006) 5490–5501 5501

[5] Sinha VR, Bansal K, Kaushik R, Kumria R, Trehan A. Poly-e-caprolactone microspheres and nanospheres: an overview. Int

J Pharma 2004;278:1–23.

[6] Pitt CG, Gratzl MM, Kimmel GL, Surles J, Schindler A. Aliphatic

polyesters. II. The degradation of poly(DL-lactide), poly(e-caprolac-tone), and their copolymers in vivo. Biomaterials 1981;2:215–20.

[7] Woodward SC, Brewer PS, Moatamed F, Schindler A, Pitt CG. The

intracellular degradation of poly(e-caprolactone). J Biomed Mater

Res 1985;19:437–44.

[8] Cha Y, Pitt CG. The biodegradability of polyester blends. Bio-

materials 1990;11:108–12.

[9] Huang M-H, Li S, Hutmacher Dietmar W, Schantz J-T, Vacanti

Charles A, Braud C, et al. Degradation and cell culture studies on

block copolymers prepared by ring opening polymerization of

e-caprolactone in the presence of poly(ethylene glycol). J Biomed

Mater Res A 2004;69:417–27.

[10] Lendlein A, Kelch S. Shape-memory polymers. Angew Chem, Int Ed

2002;41:2034–57.

[11] Lendlein A, Langer R. Biodegradable, elastic shape-memory poly-

mers for potential biomedical applications. Science 2002;296:1673–6.

[12] Ping P, Wang W, Chen X, Jing X. Poly(e-caprolactone) polyurethaneand its shape-memory property. Biomacromolecules 2005;6:587–92.

[13] Dankers PYW, Harmsen MC, Brouwer LA, Van Luyn MJA, Meijer

EW. A modular and supramolecular approach to bioactive scaffolds

for tissue engineering. Nature Mater 2005;4:568–74.

[14] Beijer FH, Sijbesma RP, Kooijman H, Spek AL, Meijer EW. Strong

dimerization of ureidopyrimidones via quadruple hydrogen bonding.

J Am Chem Soc 1998;120:6761–9.

[15] Folmer BJB, Sijbesma RP, Versteegen RM, van der Rijt JAJ, Meijer

EW. Supramolecular polymer materials: chain extension of telechelic

polymers using a reactive hydrogen-bonding synthon. Adv Mater

2000;12:874–8.

[16] Sijbesma RP, Beijer FH, Brunsveld L, Folmer BJB, Hirschberg

JHKK, Lange RFM, et al. Reversible polymers formed from self-

complementary monomers using quadruple hydrogen bonding.

Science 1997;278:1601–4.

[17] Sontjens SHM, Sijbesma RP, van Genderen MHP, Meijer EW.

Stability and lifetime of quadruply hydrogen bonded 2-ureido-4[1H]-

pyrimidinone dimers. J Am Chem Soc 2000;122:7487–93.

[18] Bosman AW, Sijbesma RP, Meijer EW. Supramolecular polymers at

work. Mater Today 2004;7:34–9.

[19] Bosman AW, Sijbesma RP. In: Ciferri A, editor. Supramolecular

polymers. 2nd ed., 2005.

[20] Gangjee A, Yu J, McGuire JJ, Cody V, Galitsky N, Kisliuk RL, et al.

Design, synthesis, and X-ray crystal structure of a potent dual

inhibitor of thymidylate synthase and dihydrofolate reductase as an

antitumor agent. J Med Chem 2000;43:3837–51.

[21] Ward IM. An introduction to the mechanical properties of solid

polymers. 2nd ed., 2004.

[22] Sung H-J, Meredith C, Johnson C, Galis ZS. The effect of scaffold

degradation rate on three-dimensional cell growth and angiogenesis.

Biomaterials 2004;25:5735–42.

[23] Sato H, Murakami R, Padermshoke A, Hirose F, Senda K, Noda I,

et al. Infrared spectroscopy studies of CH–O hydrogen bondings and

thermal behavior of biodegradable poly(hydroxyalkanoate). Macro-

molecules 2004;37:7203–13.