A modular and supramolecular approach to bioactive scaffolds for ...

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A modular and supramolecular approach to bioactive scaffolds for tissue engineering PATRICIA Y. W. DANKERS1, MARTIN C. HARMSEN2, LINDA A. BROUWER2, MARJA J. A. VAN LUYN2 AND E. W. MEIJER1* 1

Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, PO Box 513, NL-5600 MB Eindhoven, The Netherlands Department of Pathology and Laboratory Medicine, Medical Biology Section, University Medical Center Groningen, Hanzeplein 1, NL-9713 GZ Groningen, The Netherlands *e-mail: [email protected] 2

Published online: 19 June 2005; doi:10.1038/nmat1418

Bioactive polymeric scaffolds are a prerequisite for the ultimate formation of functional tissues. Here, we show that supramolecular polymers based on quadruple hydrogen bonding ureido-pyrimidinone (UPy) moieties are eminently suitable for producing such bioactive materials owing to their low-temperature processability, favourable degradation and biocompatible behaviour. Particularly, the reversible nature of the hydrogen bonds allows for a modular approach to gaining control over cellular behaviour and activity both in vitro and in vivo. Bioactive materials are obtained by simply mixing UPyfunctionalized polymers with UPy-modified biomolecules. Low-molecular-weight bis-UPy-oligocaprolactones with cell adhesion promoting UPy-Gly-Arg-Gly-Asp-Ser (UPyGRGDS) and the synergistic UPy-Pro-His-Ser-Arg-Asn (UPy-PHSRN) peptide sequences are synthesized and studied. The in vitro results indicate strong and specific cell binding of fibroblasts to the UPy-functionalized bioactive materials containing both UPy-peptides. An even more striking effect is seen in vivo where the formation of single giant cells at the interface between bioactive material and tissue is triggered.

he technique of tissue engineering has become prominent in producing new tissue using polymeric scaffolds and cells1. So-called third-generation materials have been developed, which are designed to be both resorbable and bioactive2. Tissue engineering of complex morphological structures, such as, for example, vascular grafts, require tuneable bioactive scaffolds that are intended to stimulate highly precise reactions with proteins and cells at the molecular level3. In the field of bioactivity, impressive results are disclosed on cell adhesion and spreading4,5 and on how materials can be modified to become adhesive for cells6. The most intensively studied celladhesion peptide sequence, RGD (Arg-Gly-Asp)6,7, binds to the integrin cell-surface receptors4,5. The extent of spreading depends on the amount of ligands present at the surface8. Numerous materials have been covalently functionalized with RGD peptides6. The affinity of whole extracellular matrix (ECM) proteins to integrins is significantly (>1,000 times) higher than the binding affinity of simple RGD peptides to these receptors alone9. This is explained by multivalency and the action of a synergistic PHSRN (Pro-His-Ser-Arg-Asn) peptide sequence10,11. However, it is shown that this synergistic peptide mediates cell adhesion through a similar mechanism to the RGD adhesion peptide12. The strength of adhesion depends on the distance between the RGD and PHSRN peptides13,14. Only a few examples are disclosed in which materials are covalently modified with both sequences15–22. Although covalent attachment of bioactive components to polymer backbones shows great promise, the synthetic versatility remains limited and the polymers require rather high processing temperatures. Blending polymers with bioactive components is also possible; however, these materials cannot be tuned. We propose here a new materials design by means of specific non-covalent interactions between the polymer and biomolecule that bridges the gap between covalent functionalization and simply blending of polymers with bioactive molecules. Eminently suitable for producing these novel, tuneable, bioactive materials are supramolecular polymers based on quadruple-hydrogen-bonding 2-ureido-4[1H]-pyrimidinone (UPy) moieties23–26 (Fig. 1a). These supramolecular polymers are made of repeating units held together by strong (association constant, Kass = 106–107 l mol−1) non-covalent

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© 2005 Nature Publishing Group

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Figure 1 The modular approach to bioactive supramolecular materials. a, The self-complementary hydrogen-bonding UPy moiety in a supramolecular polymer. b, The modular approach to constructing bioactive materials with various properties by simply mixing different UPy-functionalized biomolecules (green and red moieties) with UPypolymers. c, As building blocks for these materials, two UPy-functionalized peptides (UPy-GRGDS and UPy-PHSRN) were used in combination with an oligocaprolactone functionalized on both ends with UPy-units (PCLdiUPy).

hydrogen-bonding interactions23–25. The reversible nature of these hydrogen-bonding interactions (with lifetimes between 0.1–1 s) creates responsive materials and allows for a modular approach. These new materials show mechanical properties similar to macromolecules, but without losing their reversible nature26. We report here our modular approach to constructing bioactive materials with various properties, which is by simply mixing UPyfunctionalized polymers with UPy-modified biomolecules (Fig. 1b). This approach has major advantages above covalent modification of polymers, because a dynamic system is developed that can easily be tuned by mixing in different amounts of biomolecules and/or different epitopes. MODULAR DESIGN AND SYNTHESIS

We designed a box of blocks composed of various UPy-building blocks (Fig. 1c). Oligocaprolactone (biocompatible, biodegradable and FDA approved) was selected as host material and functionalized on both ends with UPy-moieties; as bioactive components the GRGDS and PHSRN peptides were selected to be modified with UPy-units. Bifunctional UPy-modified polycaprolactone (PCLdiUPy) was obtained by reaction of hydroxy telechelic low-molecular-weight polycaprolactone (PCL; Mn = 2.1 kg per mole) with the UPy-isocyanate-synthon, 2(6-isocy 2

anatohexylaminocarbonyl-amino)-6-methyl-4[1H]-pyrimidinone, in a similar manner to that described previously26. Both peptides were synthesized according to standard solid-phase peptide synthesis techniques using Fmoc-coupling chemistry. For the coupling of the UPy-units to the free amine of the last amino acid of the peptide, a method was developed to perform these reactions on the solid support. The reaction of the free amine of the protected GRGDS peptide with a 1,1΄-carbonyldiimidazole-activated27 methyl-isocytosine resulted in pure UPy-GRGDS after disconnection from the support and removal of the protecting groups. The UPy-PHSRN peptide was synthesized on the resin with the last step consisting of the reaction of the free amine with the UPy-isocyanate-synthon26, after which the UPyPHSRN peptide was deprotected and cleaved from the support. The UPy-peptides were purified using preparative reversed-phase liquid chromatography (RPLC). The compounds were characterized with a variety of techniques, including NMR, RPLC and mass spectrometry (see Supplementary Information). MATERIALS PROPERTIES

The supramolecular PCLdiUPy material showed excellent material properties. On functionalization with UPy-moieties, the properties of the oligocaprolactones changed remarkably from a waxy, brittle solid nature materials | ADVANCE ONLINE PUBLICATION | www.nature.com/naturematerials


ARTICLES (number average molecular weight Mn = 2.1 kg per mole; melting temperature Tm = 43 °C and 49 °C (melting enthalpy ΔH = 62 J g–1)) to a strong, elastic material (Mn = 2.7 kg per mole; glass-transition temperature Tg = –59 °C, Tm = 41 °C (ΔH = 15 J g–1), Tm = 64 °C (ΔH = 5 J g–1); Young’s modulus E = 49 ± 2 MPa, strain at break εbreak = 26.7 ± 1.8%, stress at break σbreak = 3.3 ± 0.1 MPa). Owing to the reversible nature of the hydrogen bonds, the PCLdiUPy material could easily be processed by different techniques into several scaffolds (Fig. 2) varying from films and fibres to meshes and grids. Films were made by solvent casting or compression moulding. Melt spinning and electrospinning have been used to make fibres and meshes. Grids consisting of filaments with a width down to approximately 220 µm were produced by fused deposition modelling (FDM)28. The dynamic nature of our supramolecular materials gives rise to lowmelt viscosity, which makes processing of these new polymers at temperatures just below 80 °C possible. The biocompatibility of the UPy-unit was tested in vitro with two water-soluble model compounds (see Supplementary Information). These viability tests strongly indicate that the UPy-moiety is not toxic and thus biocompatible. Subcutaneous implantation of PCLdiUPy films in rats confirms these findings by showing no priming of the immune system (see later in Fig. 5). PCLdiUPy hardly degrades in buffer but becomes more crystalline in time. The degradation of PCLdiUPy can be enzymatically tuned with lipases in vitro (see Supplementary Information). Even when subcutaneously implanted in vivo, hardly any degradation can be detected. However, supramolecular bioactive PCLdiUPy shows interesting degradation behaviour in vivo (see below). Several polymer–peptide films made in two different ways were used for the in vitro and in vivo cellular tests that were performed for this study (see Methods). The surface quality of second set of films (method two) is better than that of the first set of films (method one). The exact position of the peptides, the binding of the peptides to the polymers and the distribution of the peptides throughout the polymer films are not exactly known. Extraction experiments on the second set of films show (see Supplementary Information) that dissolution of GRGDS without a UPy-moiety proceeds extremely fast in water; within five minutes all of the peptide is dissolved. The extraction of UPy-GRGDS with water from the polymer film is a slow process indicating the importance of the UPy-unit for the tuneable but dynamic binding of the peptide to the polymer. Contact-angle measurements on the first set of films before cell seeding indicate the presence of peptides at the surface, because the angle of the blend with UPy-GRGDS (48 ± 3°) or with GRGDS (51 ± 4°) is lower than for the pure PCLdiUPy film (63 ± 5°). All of these experiments are in agreement with the dynamic nature of our UPy-based polymers and oligopeptides. The UPypeptides dissolve slowly in water, due to the strong, but reversible binding to the base polymer. The binding strength is dependent on the position of the UPy-peptide: from high Kass in the bulk of the film to low Kass in water. Factors such as morphology of the film and hydrophobicity of the UPy-peptide determine the lifetime of the supramolecular bond between the bioactive peptide and the UPy-polymer at the polymer–water interface. CELL ADHESION AND SPREADING

Mouse 3T3 fibroblast cells (5 × 104 cells cm–2) were cultured by method one on different supramolecular polymer–peptide blends for two days in the absence of FBS (fetal bovine serum) to prevent cell adhesion by absorbed serum proteins (Fig. 3). PCLdiUPy polymers were mixed with UPy-GRGDS (1), with UPy-PHSRN

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Figure 2 Processability of the supramolecular UPy-materials. The PCLdiUPy polymer was processed into several scaffolds; films, fibres, meshes (SEM pictures; Philips XL30 FEG SEM) and grids by FDM.

(2) and with both UPy-GRGDS and UPy-PHSRN (3). In all cases 4 mol% of peptide was mixed with the PCLdiUPy solution before preparing the films. The bare PCLdiUPy polymer (4) is shown as control (Fig. 3). After seeding of the cells, the samples were followed over time by optical microscopy. Aspecific adhesion but hardly any cell spreading was already visible after three hours on all samples, even on all controls (see Supplementary Information). However, after one day, additional cell spreading and the highest degree of cell adhesion was observed for cells seeded on blend 3, which might indicate the possible synergistic effect of the two UPypeptides (see Supplementary Information for statistical analysis on the extent and morphology of the cell adhesion). On the film of 1 some cells adhered and spread after one day, but less efficient lythan on blend 3. Also, fewer cells spread on mixture 2 after one day. This has been proposed to be due to the fact that PHSRN is a synergistic sequence10, although it is also shown that this synergistic peptide mediates cell adhesion through a similar mechanism to the RGD adhesion peptide12. These findings for the different films remained unchanged even after two days (Fig. 3). Similar results were found for the second set of bioactive supramolecular materials (see Supplementary Information). If different concentrations of the UPy-GRGDS peptides, 1, 2 or 4 mol%, were swollen on the polymer films made according to method two, hardly any differences in cell adhesion and spreading were observed between the samples. Controls with peptides without a UPy-unit showed that they were not able to induce cell adhesion and spreading, which was also shown for polymer 4 (Fig. 3), confirming the extraction experiments in which the peptides without a UPy-moiety rapidly diffuse out of the film. Moreover, the presence of water-soluble GRGDS peptides in the medium can influence the binding of the cells to bioactive materials as shown in an inhibition experiment where 3T3 fibroblasts were first incubated with GRGDS peptides before seeding on blend 3 (Fig. 4a). Without incubation, the cells adhered and spread out, however, after incubation of the cells with the soluble GRGDS peptides hardly any adhesion and spreading could be detected after one day. This indicates that the cell binding might be integrin-mediated. The second set of films was incubated in medium without FBS for three hours before seeding of the cells on the polymers to test



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Figure 3 Cell adhesion and spreading in vitro. Fibroblast cell (5 × 104 cells cm–2) adhesion and cell spreading on different drop-cast films of mixtures of PCLdiUPy with UPy-GRGDS (1), PCLdiUPy with UPy-PHSRN (2), PCLdiUPy with both UPy-GRGDS and UPy-PHSRN (3) and PCLdiUPy alone (4), after two days of cell culturing in the absence of FBS. In all cases 4 mol% of peptide was mixed with the PCLdiUPy. The cells were visualized on the polymer films with optical microscopy. Scale bars represent 100 µm.

the stability of the bioactive materials. After this incubation step, the samples were washed twice with a phosphate-buffered saline solution and cells were cultured on these films for one day, again in the absence of FBS. This resulted in similar adhesion and spreading patterns as shown before (see Fig. 3 and Supplementary Information). All these experiments—with and without peptides—show that UPy-peptides are really necessary to promote cell adhesion and spreading in vitro due to the dynamic binding of the peptides to the supramolecular material. Despite the reversible nature of the binding, the stability of the bioactive polymer is high enough to function as bioactive material. CELL-BINDING STRENGTH

The cells cultured on blend 3 (made according to method two) without FBS look similar to cells cultured on blend 3 or on the bottom of a polystyrene (PS) culture dish in the presence of FBS after one day of incubation (Fig. 4b). These results indicate that the peptides facilitate cell adhesion and spreading in the same way as ECM proteins in FBS. Differences, however, can be found in cellbinding-strength experiments using trypsin (Fig. 4b). After 30 seconds of incubation with trypsin–EDTA, the cells on blend 3 with FBS and on the PS with FBS were completely detached (Fig. 4b). These cells were subsequently washed off the plates leaving behind some floating single cells. In contrast, even after 30 minutes of incubation with trypsin–EDTA the cells on blend 3 in the absence of FBS were still adhered and hardly any floating cells were observed. After removal of the trypsin–EDTA and washing of the fibroblasts, they could spread again on blend 3 without FBS when incubated for one additional day (Fig. 4b), suggesting that the UPypeptides can act in a reversible fashion. These trypsin experiments indicate that the new supramolecular materials approach affords strong binding, but that the mechanism of binding is sensitive for competitive ECM proteins. IN VIVO BEHAVIOUR

Owing to the dynamic behaviour of the supramolecular polymer and the UPy-functionalized oligopeptides under aqueous conditions, it is difficult to quantify the cell experiments on surfaces. Cell adhesion has been shown in vitro but it remains unclear whether these materials are functional under physiological conditions. Therefore, we have studied this system in vivo by subcutaneous implantation in male Albino Oxford rats (Fig. 5). Solution-cast polymer films 4

were made by a similar method to that described for the first set of materials, except for the fact that the films were much thicker, which is required for in vivo observations. The bare PCLdiUPy 4 was compared with blend 3. Both materials were slightly opaque and blend 3 seemed to be rather stiff and somewhat brittle. The differences observed after in vivo implantation between both supramolecular materials are striking. At day five, the cellular infiltration was very mild for polymer 4 and a small fibrous capsule had formed, reflecting the inert and adhesive characteristics of this material (Fig. 5). However, in the case of blend 3, vascularization and infiltration of macrophages was observed (Fig. 5), which might be due to the presence of the peptides that could recruit cells through integrin binding. Another remarkable difference was the fact that in the case of blend 3 after five days, large giant cells were already budding into the material from the interface (Fig. 5), which indicates that the UPy-GRGDS and UPy-PHSRN peptides may not only play a part in the signalling and infiltration of macrophages, but also in their fusion to giant cells. Increased giant cell formation was also detected when polymer polyethyleneglycol-based networks grafted with RGD and PHRSN peptides were subcutaneously cage implanted21. It was indicated that both peptides are important in modulating the macrophage function21. Giant cells were not detected in polymer 4 and the cellular response was negligible up to 42 days. However, the tissue response for blend 3 became even more active after 10 days, and degradation of the polymer was shown by phagocytotic activity of giant cells present in the surrounding tissue. The giant cells at the interface still did not show any phagocytotic behaviour up to 42 days, although ongoing degradation was observed after 42 days (Fig. 5). At this time point macrophage infiltration was no longer observed and the surrounding tissue became very inactive. These results show that the peptides used are able to induce signalling of cells and induction of angiogenesis. OUTLOOK

The exact mechanism of binding and spreading of our UPymaterials has still to be elucidated. Although the UPy–UPy association constant in water is low, the hydrophobic shielding of this bond in the upper layer of the polymer film makes this binding strong but dynamic. The leakage of the peptides out of the films is strongly reduced by the UPy-moiety and can be tuned by the attachment of different UPy-groups. An interesting option for the novel supramolecular materials is their ability to adapt their structure to the cells presented. It is proposed that they are not only bound but can also dynamically ‘move’ over the surface. nature materials | ADVANCE ONLINE PUBLICATION | www.nature.com/naturematerials


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Figure 4 Specific cell binding and reversibility of the spreading mechanism. a, Inhibition experiment. Fibroblast cells on synergistic blend 3 containing PCLdiUPy with both UPy-GRGDS and UPy-PHSRN in the absence of FBS adhere and spread without pre-incubation with soluble GRGDS peptides (‘no soluble GRGDS’), but hardly any adhesion and spreading can be detected after pre-incubation of the cells with soluble GRGDS peptides (‘soluble GRGDS’). The cells were visualized on the polymer films with optical microscopy after one day of culturing at 37 °C. Scale bars represent 100 µm. b, Trypsin experiment. The cells adhere and spread after one day of incubation at 37 °C both on synergistic blend 3 in the absence and presence of FBS and on polystyrene in the presence of FBS (PS + FBS) (‘first day’). Only the cells on synergistic blend 3 cultured in the absence of FBS were still adhered after trypsin incubation, removal of the trypsin solution and washing of the cells (‘trypsin’). The cells were able to spread again on this synergistic blend 3 after incubation at 37 °C (‘second day’). The cells were visualized on the polymer films with optical microscopy. Scale bars represent 100 µm.

The role of this dynamic character of supramolecular films to cell adhesion is the subject of current investigations. In vivo experiments showed that the oligopeptides have an important influence on the signalling of cells in the surrounding tissue and on the functioning of cells around the material. These

materials will be subjected to more intensive studies to elucidate the role of the different peptides on the supramolecular polymer properties and the presence of the UPy-unit in vivo. Above all, the beauty of the system is the possibility to easily change to various polymers and biofunctionalities; even proteins that can regulate



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Figure 5 In vivo behaviour of the supramolecular bioactive materials. Solution-cast supramolecular PCLdiUPy films without peptides (4) and with UPy-GRGDS and UPy-PHSRN peptides (3) were subcutaneously implanted in male Albino Oxford rats. The films were explanted with the surrounding tissue after 5, 10 and 42 days. These peptides have an important influence on the signalling of the cells in the surrounding tissue. Giant cell formation is shown at the interface of material 3. Vascularization is also enhanced for material 3. After 42 days the surroundings become inactive for both blends. The samples were stained with toluidine blue for histological examination. The magnifications are 200 times. The material is indicated with m. The fibrous capsule is shown with c. Blood vessels are indicated with v. The giant cells that are budding into the material from the interface are indicated with an asterisk (*). The first enlargement (blend 3, day 5) shows a strip of blood vessels, indicated with circles. The second enlargement (blend 3, day 10) shows phagocytotic giant cells that contain polymer degradation products, in the surrounding tissue. The magnification of the enlargements are 400 times.

and stimulate all kinds of intra- and intercellular processes might be modified with those UPy-moieties. Now we anticipate that the road is open to expand this strategy to incorporate these different bioactive molecules to get exquisite control over highly sophisticated scaffold architectures. METHODS GENERAL METHODS The general materials and instrumentation used in this work are described in the Supplementary Information.

BUILDING BLOCKS The PCLdiUPy polymer and the UPy-GRGDS and UPy-PHSRN peptides were synthesized and characterized as described in the Supplementary Information. The PCLdiUPy material was processed by different techniques into several scaffolds (see Supplementary Information).

PREPARATION OF THE FILMS Two methods were used to prepare bioactive supramolecular UPy films. In the first set of films (method one), the peptides were dissolved in tetrahydrofuran (THF) with 10% water and the PCLdiUPy polymers were dissolved in THF. A bioactive blend with 4 mol% of each peptide was produced by mixing both solutions. The resulting mixture was drop-cast on glass cover slips (diameter = 1.5 cm; 1 × 10–4 mmol peptide and 2.4 × 10–3 mmol polymer per cover slip) for the in vitro experiments or in Petri dishes for the in vivo experiments. Most of the time, a slight precipitation was visible. In the second set of films (method two) the polymer solution in THF was first drop-cast on the glass cover slips. Subsequently, the peptide solution (4 mol% of each peptide in THF with 10% water) was drop-cast on the dried polymer film. The blends on the glass cover slips were dried in vacuo for 2–3 days at 35–40 °C. The samples were sterilized under UV for at least three hours before use.

CELL GROWTH 3T3 mouse fibroblasts (polymerase chain reaction, PCR, on cDNA made from the RNA of these cells showed the presence of mRNA of the α5, β1, αv and β3 integrin subunits) were cultured on a 1:1 mixture of Ham’s F-12 and Dulbecco’s Modified Eagle’s Medium with 10% FBS. They were cultured in a humidified incubator at 37 °C and 5% CO2. Before seeding of the cells on the materials, they were washed twice with PBS solution. Then they were trypsinized with a trypsin–EDTA solution (it was checked with fluorescence-activated cell sorting (FACS) measurements that cells contain

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the α5β1 integrins irrespective of treatment of the cells with trypsin–EDTA or with EDTA solution), washed with PBS and counted after trypan-blue staining in a Neubauer counting chamber. The cells were seeded in the culture medium (with or without FBS supplemented, as indicated) on the films. The passage of the cells was always between 10 and 80, and the viability of the cells was always above 97%.

IN VITRO CELL EXPERIMENTS Cell-adhesion and cell-spreading experiments. 3T3 mouse fibroblasts (5 × 104 cells cm–2) were seeded on the cover slips with the supramolecular bioactive materials or with the bare PCLdiUPy, on the bottom of a PS culture dish and on glass in 200 µl medium (with or without FBS, as indicated). They were incubated for five minutes at room temperature, after which 1 ml medium (with or without FBS, as indicated) was added. During one or two days of culturing in a humidified incubator at 37 °C and 5% CO2, they were studied with optical microscopy (Zeiss Axiovert 25 microscope with a DSC-S75 Sony digital still camera). Inhibition experiments. 3T3 mouse fibroblasts (4 × 105 cells ml–1 medium without FBS) were incubated at room temperature for 15 minutes with soluble GRGDS peptides (0.3 mM in medium without FBS). After this incubation step the cells (6 × 104 cells cm–2) were seeded on blend 3 in 250 µl medium without FBS. In the case of the control, the cells (6 × 104 cells cm–2) were not pre-incubated with these soluble GRGDS peptides. After seeding of the cells, they were incubated for five minutes at room temperature. Then 1 ml medium without FBS was added. After one day of culturing in a humidified incubator at 37 °C and 5% CO2, they were studied with optical microscopy. Cell binding strength and cell-spreading reversibility experiments. Trypsin experiments were performed after one day of culturing the 3T3 mouse fibroblasts (5 × 104 cells cm–2) at 37 °C and 5% CO2. The cells on blend 3 in the absence of FBS or in the presence of FBS and the cells on the bottom of the polystyrene culture dish in the presence of FBS (PS + FBS) were incubated in a trypsin-EDTA solution at room temperature for 30 seconds to 30 minutes. After removal of the trypsin-EDTA solution the cells were washed twice with PBS solution. Cell-culture medium without FBS was added in the case of blend 3 (that was incubated without FBS) and the remaining cells were incubated again in a humidified incubator at 37 °C and 5% CO2 for 1 day. During the whole process the cells were followed with optical microscopy.

IN VIVO IMPLANTATIONS Solution cast PCLdiUPy films (diameter = 6 mm, thickness = approximately 0.4 mm) without (material 4) and with 4 mol% of UPy-GRGDS and 4 mol% of UPy-PHSRN (blend 3) were subcutaneously implanted in duplicate into male Albino Oxford rats. The implants with the surrounding tissue were explanted after 5, 10 and 42 days of implantation and were embedded in plastic (Technovit 7100 cold curing resin based on hydroxyethylmethacrylate (HEMA), Kulzer HistoTechnik). The samples were stained with toluidine blue for histological examination with optical microscopy (Leica DMLB microscope with Leica DC300 camera).



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Acknowledgements We would like to thank Jolanda Spiering for the synthesis of the functionalized polymer in large quantities, Hans Adams for the help with the peptide synthesis, Gaby van Gemert and Henk Janssen for synthesizing the water-soluble UPy-molecules, Rob Hermans for the help with the toxicity tests, Ralf Bovee for the help with the preparative RPLC, Nico Kamperman and Maarten de Graauw for producing the FDM scaffolds, Cláudia Vaz for making the electrospun scaffold, Linda van Beek for spinning the fibres, Sagitta Peters for the degradability studies and Guido Krenning for the great help with the PCR and FACS study. Finally, we like to thank the many fruitful discussions with Rint Sijbesma, Nico Sommerdijk, Carlijn Bouten and Frank Baaijens. This work is supported by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO). Correspondence and requests for materials should be addressed to E. W. M. Supplementary Information accompanies the paper on www.nature.com/naturematerials.

Competing financial interests The authors declare that they have no competing financial interests.



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