Three-Dimensional Biodegradable Structures Fabricated by Two

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Three-Dimensional Biodegradable Structures Fabricated by Two-Photon Polymerization Frederik Claeyssens,*,† Erol A. Hasan,‡ Arune Gaidukeviciute,§ Demetra S. Achilleos,§,| Anthi Ranella,§ Carsten Reinhardt,§,⊥ Aleksandr Ovsianikov,⊥ Xiao Shizhou,⊥ Costas Fotakis,§,# Maria Vamvakaki,§,| Boris N. Chichkov,§,⊥ and Maria Farsari*,§ Engineering Materials Department, Biomaterials and Tissue Engineering Group, Kroto Research Institute, UniVersity of Sheffield, Broad Lane, Sheffield, S3 7HQ, United Kingdom, Department of Chemistry, UniVersity of Cambridge, Cambridge, CB2 1EW, United Kingdom, Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), P.O. Box 1527, 711 10 Heraklion, Crete, Greece, Department of Materials Science and Technology and Department of Physics, UniVersity of Crete, Greece, and Laser Zentrum HannoVer e.V., Germany ReceiVed NoVember 16, 2008. ReVised Manuscript ReceiVed December 15, 2008 Two-photon polymerization has been employed to fabricate three-dimensional structures using the biodegradable triblock copolymer poly(ε-caprolactone-co-trimethylenecarbonate)-b-poly(ethylene glycol)-b-poly(ε-caprolactoneco-trimethylenecarbonate) with 4,4′-bis(diethylamino)benzophenone as the photoinitiator. The fabricated structures were of good quality and had four micron resolution. Initial cytotoxicity tests show that the material does not affect cell proliferation. These studies demonstrate the potential of two-photon polymerization as a technology for the fabrication of biodegradable scaffolds for tissue engineering.

Introduction Tissue engineering applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ.1 Creating an engineered tissue typically constitutes of three consecutive steps: (i) identifying a material suitable for the fabrication of a scaffold for a specific tissue engineering application, (ii) structuring of the scaffold material, and (iii) cell seeding into the scaffold for cell culturing in Vitro or in ViVo. A matter of considerable interest recently is the creation and structuring of biocompatible materials to enable the integration of living cells. The choice of the scaffold for cell cultivation can greatly influence the attachment, migration, and proliferation of cells. Scaffold materials that are not rejected by the body upon implantation can be selected from metals, ceramics, synthetic polymers, and biopolymers. Specific tissue engineering applications demand different materials properties; for example, bone replacements need strong and wear resistant materials such as metals and ceramics, while soft tissue replacements can be made of polymeric and biological materials.2-4 In soft tissue engineering, the primary function of the scaffold structure is to provide a micro- and nanostructured three-dimensional (3D) environment for the cells to migrate and to proliferate in. The specific properties of the cell scaffold have to be tuned for each tissue engineering * Corresponding authors. E-mail: [email protected] (M.F.); [email protected] (F.C.). † University of Sheffield. ‡ University of Cambridge. § Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH). | Department of Materials Science and Technology, University of Crete. ⊥ Laser Zentrum Hannover e.V. # Department of Physics, University of Crete.

(1) Langer, R.; Vacanti, J. P. Science 1993, 260, 920–926. (2) Hutmacher, D. W.; Schantz, T.; Zein, I.; Ng, K. W.; Teoh, S. H.; Tan, K. C. J. Biomed. Mater. Res. 2001, 55(2), 203–216. (3) Jeong, S. I.; Kim, B. S.; Lee, Y. M.; Ihn, K. J.; Kim, S. H.; Kim, Y. H. Biomacromolecules 2004, 5(4), 1303–1309. (4) Risbud, M. V.; Sittinger, M. Trends Biotechnol. 2002, 20(8), 351–356.

application, since the 3D environment surrounding the cells provides instructive cues needed to maintain cell phenotype and behavior. Once the cells of the engineered tissue have built their own connective tissue, the scaffold becomes redundant. Therefore, biomaterials that are bioresorbable and biodegradable on a similar time scale as that of the production of the extracellular matrix of the engineered tissue are preferred. Micron-sized topography has been shown to play an essential role in determining cell adhesion, and surface-bound characteristics influence in this way prominent cellular functions such as survival, proliferation, differentiation, migration, or mediator release.5 In particular, 3D cell cultures offer a more realistic micro- and local-environment where the functional properties of cells can be observed and manipulated.6,7 An important factor in the production of working tissue engineering scaffolds is the ability of a reproducible and controlled method of nanostructuring. A versatile class of scaffold production techniques which enable the fabrication of tailored structures directly from computer data via computer aided design/computer aided manufacturing (CAD/ CAM) are solid-free-form (SFF) fabrication techniques.8 A number of SFF techniques have already been implemented and commercialized, including stereolithography,9-11 laminated object manufacturing,12,13 selective laser sintering,14,15 and fused deposition modeling.16 (5) Roach, P.; Eglin, D.; Rohde, K.; Perry, C. C. J. Mater. Sci.: Mater. Med. 2007, 18(7), 1263–1277. (6) Zhang, S. G.; Gelain, F.; Zhao, X. J. Semin. Cancer Biol. 2005, 15(5), 413–420. (7) Abbott, A. Nature 2003, 424, 870–872. (8) Hollister, S. J. Nat. Mater. 2005, 4(7), 518–524. (9) Cooke, M. N.; Fisher, J. P.; Dean, D.; Rimnac, C.; Mikos, A. G. J. Biomed. Mater. Res. 2003, 64B(2), 65–69. (10) Dhariwala, B.; Hunt, E.; Boland, T. Tissue Eng. 2004, 10(9-10), 1316– 1322. (11) Fisher, J. P.; Vehof, J. W. M.; Dean, D.; Waerden, J. v. d.; Holland, T. A.; Mikos, A. G.; Jansen, J. A. J. Biomed. Mater. Res. 2002, 59, 547–556. (12) Mikos, A. G.; Sarakinos, G.; Leite, S. M.; Vacanti, J. P.; Langer, R. Biomaterials 1993, 14(5), 323–330. (13) Gleghorn, J. P.; Lee, C. S. D.; Cabodi, M.; Stroock, A. D.; Bonassar, L. J. J. Biomed. Mater. Res., Part A 2008, 85A(3), 611–618. (14) Tan, K. H.; Chua, C. K.; Leong, K. F.; Cheah, C. M.; Cheang, P.; Abu Bakar, M. S.; Cha, S. W. Biomaterials 2003, 24(18), 3115–3123.

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Figure 1. Chemical structure of the employed photopolymer.

3D laser nonlinear lithography has been demonstrated as a technology for the fabrication of 3D structures with high resolution capabilities.17-19 The technique is based on the phenomenon of two-photon polymerization (2PP). When the beam of an ultrafast infrared laser is tightly focused into the volume of a photosensitive material, the polymerization process can be initiated by twophoton absorption within the focal region. By moving the laser focus three-dimensionally through the photosensitive material, arbitrary 3D structures can be fabricated.20 Recently, 2PP was explored for use in the construction of permanent scaffolds using the photosensitive organic-inorganic hybrid sol-gel ORMOCER and the negative photoresist SU8.21-24 In this paper, we report the fabrication by 2PP of 3D structures consisting of the triblock copolymer poly(ε-caprolactone-cotrimethylenecarbonate)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-trimethylenecarbonate) (PCL-co-PTMC)-b-PEG-b(PCL-co-PTMC). Polycaprolactone-based scaffolds have been studied extensively in tissue engineering applications.2,25 Moreover, previous studies on this triblock copolymer have shown that not only it is biocompatible and biodegradable, but also it degrades on a similar time scale as tissue formation.26,27

Results and Discussion Polymer Microstructuring. The structure of the polymer employed in this work is shown in Figure 1. Figure 2a shows a scanning electron microscopy (SEM) picture of a structure built by 2PP at 500× magnification. The structure consists of six step-in squares and five horizontal/vertical lines, which serve as cell dividers. To fabricate the structure, the same layer was repeated eight times and the linear stage moved the sample lower by 6 µm after each layer. A scanning speed of 50 µm/s was employed, requiring a minimum power of 30 mW (measured before the objective) to initiate two-photon polymerization. A side view of the same structure is shown in Figure 2b, indicating that the height of the structure is 50 µm. A moderate distortion can be clearly seen due to polymer shrinkage. Figure 2c shows (15) Chua, C. K.; Leong, K. F.; Tan, K. H.; Wiria, F. E.; Cheah, C. M. J. Mater. Sci.: Mater. Med. 2004, 15(10), 1113–1121. (16) Too, M. H.; Leong, K. F.; Chua, C. K.; Du, Z. H.; Yang, S. F.; Cheah, C. M.; Ho, S. L. Int. J. AdV. Manuf. Technol. 2002, 19(3), 217–223. (17) Cumpston, B. H.; Ananthavel, S. P.; Barlow, S.; Dyer, D. L.; Ehrlich, J. E.; Erskine, L. L.; Heikal, A. A.; Kuebler, S. M.; Lee, I. Y. S.; McCordMaughon, D.; Qin, J. Q.; Rockel, H.; Rumi, M.; Wu, X. L.; Marder, S. R.; Perry, J. W. Nature 1999, 398(6722), 51–54. (18) Jun, Y.; Nagpal, P.; Norris, D. J. AdV. Mater. 2008, 20, 606–610. (19) Bhuian, B.; Winfield, R. J.; O’Brien, S.; Crean, G. M. Appl. Surf. Sci. 2006, 252, 4845–4849. (20) Sun, H.-B.; Kawata, S. Two-Photon Photopolymerization and 3D Lithographic Microfabrication. In NMR. 3D analysis. Photopolymerization; Fatkullin, N., Ed.; Springer: Berlin/Heidelberg, 2004; Vol. 170, pp 169-273. (21) Narayan, R. J.; Jin, C. M.; Doraiswamy, A.; Mihailescu, I. N.; Jelinek, M.; Ovsianikov, A.; Chichkov, B.; Chrisey, D. B. AdV. Eng. Mater. 2005, 7(12), 1083–1098. (22) Doraiswamy, A.; Jin, C.; Narayan, R. J.; Mageswaran, P.; Mente, P.; Modi, R.; Auyeung, R.; Chrisey, D. B.; Ovsianikov, A.; Chichkov, B. Acta Biomater. 2006, 2(3), 267–275. (23) Ovsianikov, A.; Chichkov, B.; Adunka, O.; Pillsbury, H.; Doraiswamy, A.; Narayan, R. J. Appl. Surf. Sci. 2007, 253(15), 6603–6607. (24) Ovsianikov, A.; Schlie, S.; Ngezahayo, A.; Haverich, A.; Chichkov, B. N. J. Tissue Eng. Regener. Med. 2007, 1, 443–449. (25) Kweon, H.; Yoo, M. K.; Park, I. K.; Kim, T. H.; Lee, H. C.; Lee, H. S.; Oh, J. S.; Akaike, T.; Cho, C. S. Biomaterials 2003, 24(5), 801–808. (26) Mizutani, M.; Matsuda, T. J. Biomed. Mater. Res. 2002, 61(1), 53–60. (27) Mizutani, M.; Matsuda, T. Biomacromolecules 2002, 3, 249–255.

a 1900× magnification picture of the structure, from which a resolution of ∼4 µm is inferred. Additionally, Figure 2d presents a 5 × 5 array of free-standing hollow cylinders, having outer diameters of 50 µm and inner diameters of 30 µm. The strands of material observable in between some of the free-standing cylinders are remnants of nonpolymerized prepolymer. As shown in Figure 3a, it is possible to fabricate free-standing objects providing a real three-dimensional structure using this material. In this case, the scanning speed was again 50 µm/s and the laser power 30 mW. A 1600× magnification detail of the same structure can be seen in Figure 3b. The structure retains its shape and controlled porosity, but it suffers from distortion, most likely due to polymer shrinkage during photopolymerization and material softness. The size of the structures fabricated using 2PP is limited by the field of view of the microscope objective used to focus the fs laser beam. This limitation can be overcome by (i) using large travel x-y motorized stages, to move the sample rather than the laser beam, and (ii) using a step-and-repeat strategy, where the galvanometric scanner is used to create small areas of structures and x-y stages are used to move the sample, so that the scanning can be repeated. An example of this approach is shown in Figure 4, where a two-dimensional scaffold structure was fabricated using this strategy. Cytotoxicity Test. As mentioned above, the photosensitive copolymer used in this study has been studied earlier for its biocompatibility and biodegradability.26,27 We have carried out some initial cytotoxicity tests to investigate whether the copolymer structures fabricated in this work will interfere with the process of cell division and thus result in a reduction of the cell growth rate. Balb/c 3T3 NIH cells (an established fibroblast cell line) were grown onto spin-coated thin films of photopolymerized material as well as on glass surfaces (control culture) for 2-7 days. To assess the cytotoxicity of the copolymer on the fibroblasts, cell viability was measured using a Live-Dead Cell Staining Kit. In this assay, a cell-permeable green fluorescent dye was utilized to stain live cells. Dead cells can be easily stained by propidium iodide (PI), a cell nonpermeable red fluorescent dye. Stained live and dead cells were visualized by fluorescence microscopy using a band-pass filter. After 2-7 days of cultivation, a confluent monolayer (10× magnifications) of well-defined 3T3 mouse fibroblast cells was observed exhibiting cell-to-cell contact as shown in Figure 5. This figure strongly indicates that fibroblast cells can attach and divide on the surface of the polymer as effectively as on the glass coverslips. These preliminary experiments show that the copolymer did not affect cell proliferation.

Conclusions A polycaprolactone-based triblock copolymer was synthesized and was further utilized to build three-dimensional structures by 2PP with 4 µm resolution. Preliminary cytotoxicity tests carried out on the photopolymerized materials showed that they do not affect cell proliferation. This study indicates that two-photon polymerization presents an interesting fabrication route for biodegradable microstructured materials to be used as tissue engineering scaffolds.

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Figure 2. Top view (a), side view (b), and detail (c) of a 3D structure fabricated using two-photon polymerization. Large cylindrical structures (d).

Experimental Section Polymer Synthesis. Polyethylene glycol (PEG) with Mn ) 400 g/mol (Aldrich), ε-caprolactone (CL) (Aldrich), Trimethylene carbonate (TMC, 1,3-dioxanone-2) (Boehringer KG, Ingelheim/ Rhein, Germany), and tin 2-ethylhexanoate (Sn(C8H15O2)2) (Aldrich) were used for polymer synthesis. Methacryloyl chloride (Fluka) and triethylamine (TEA) (Fluka), used for the modification of the hydroxyl end groups of the polymer, were freshly distilled before use. 4,4′bis(diethylamino)benzophenone (Sigma-Aldrich) was used as the photoinitiator for two-photon polymerization.19,28,29 All solvents were purchased from Aldrich and were used without further purification. PEG 400 (10 g, 0.025 mol) was transferred to a two-necked flask and dried via a Dean-Stark apparatus using toluene as azeotropic solvent. Next, TMC (10 g, 0.1 mol), CL (11.2 g, 0.1 mol), and Sn(C8H15O2)2 (0.004 g, 1 µmol) were added to the freshly dried PEG under a nitrogen atmosphere. The mixture was stirred for 16 h at (28) Ovsianikov, A.; Gaidukeviciute, A.; Chichkov, B. N.; Oubaha, M.; MacCraith, B. D.; Sakellari, I.; Giakoumaki, A.; Gray, D.; Vamvakaki, M.; Farsari, M.; Fotakis, C. Laser Chem. 2008, 2008, 493059. (29) Ovsianikov, A.; Viertl, J.; Chichkov, B.; Oubaha, M.; MacCraith, B.; Sakellari, I.; Giakoumaki, A.; Gray, D.; Vamvakaki, M.; Farsari, M.; Fotakis, C. ACS Nano 2008, 2(11), 2257–2262.

180 °C under N2. The copolymer was isolated by precepitation in methanol and dried under vacuum. The final product was characterized by 1H NMR spectroscopy: δ 1.30 (2H, -OOC-CH2CH2-CH2-CH2-CH2-) 1.55 (4H, -OOC-CH2-CH2-CH2CH2-CH2-), 1.93 (2H, -OOCO-CH2-CH2-CH2-), 2.30 (2H, OOC-CH2-CH2-CH2-CH2-CH2), 3.50 (4H, -CH2-CH2-O-), 4.03 (2H, -OOC-CH2-CH2-CH2-CH2-CH2-), 4.11 (4H,OOCO-CH2-CH2-CH2-), 4.30 (1H, -OOC-CH2-CH2-CH2CH2-CH2-OH), 4.55 (1H, -OOCO-CH2-CH2-CH2-OH). The polymer composition as determined by NMR was 69-14.5-16.5 mol % PEO-CL-TMC. The hydroxyl end groups of the polymer (Mn ) 1050 g/mol) were further modified with methacryloly chloride to produce a polymer carrying polymerizable methacrylate moieties. The reaction was carried out in dry THF. In a typical reaction, the prepolymer (5 g, 4.8 mmol) was transferred into a 250 mL flask and dried under reduced vacuum for 0.5 h. Subsequently, dry THF (170 mL) was injected via a syringe into the reaction flask under nitrogen. Next, TEA (10.6 mL, 0.08 mol) was added, followed by the dropwise addition of methacryloly chloride (3.7 mL, 0.04 mol). The reaction was allowed to proceed for 1 day at RT. The triethylamine salt produced was removed by filtration under nitrogen while the excess amount of methacryloly chloride and TEA and the solvent were removed under reduced pressure. Finally, the product was dried and

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Figure 3. Overview (a) and detail (b) of a 3D structure.

Figure 4. Cell structure fabricated using a “step-and-repeat” building strategy.

characterized by 1H NMR (500 MHz, CDCl3): δ 1.38 (2H, -OOC-CH2-CH2-CH2-CH2-CH2-), 1.64 (4H, -OOC-CH2-

Claeyssens et al. CH2-CH2-CH2-CH2-), 1.93 (3H, (CH3CdCH2), 2.0 (2H, -OOCO-CH2-CH2-CH2-), 2.30 (2H, -OOC-CH2-CH2CH2-CH2-CH2-), 3.63 (4H, -CH2-CH2-O-), 4.13 (2H, -OOC-CH2-CH2-CH2-CH2-CH2-), 4.22 (4H, -OOCOCH2-CH2-CH2-), 5.56 and 6.10 (1H, -(CH3)CdCH2). The disappearance of the peaks attributed to the protons of the hydroxyl end groups in the 1H NMR spectrum of the product verified the complete modification of the hydroxyl end groups to the methacrylate functionalities. To prepare the photosensitive mixture, 0.5 g of the polymer carrying the methacrylate functionalities was dissolved in 250 µL diethyl ether, followed by the addition of the 4,4′-bis(diethylamino)benzophenone photoinitiator (2 wt% to the polymer). A small droplet was deposited on a microscope coverslip and was placed in a vacuum oven for ∼5 h to evaporate the solvent. The material remained liquid after baking, so the sandwich-sample format was used, as described previously.30 The spacer between the two coverslips was 100 µm thick. To promote sample adhesion, the coverslips were bathed in a dilute solution of 3-(trimethyoxysilyl)propyl methacrylate (MAPTMS, 99%, Polysciences Inc., 40 mM in CH2Cl2) before use. 2PP Experimental Setup. The procedure for the fabrication of 3D microstructures by multiphoton polymerization has been described by several groups.31-34 In this work, the light source used was a Ti:sapphire femtosecond laser operating at 800 nm with a repetition rate of 75 MHz (Femtolasers Fusion). This source is a compact diode-pumped fentosecond laser oscillator with integrated dispersive mirrors that precompensate for the beam delivery and focusing optics to achieve sub-20 fs duration pulses into the sample. The maximum output power of the oscillator is 450 mW. The photopolymerized structure was generated using an x-y galvanometric mirror digital scanner (Scanlabs Hurryscan II) controlled by SAMLight (SCAPS) software. The scanner was adapted to accommodate a high numerical aperture focusing microscope objective lens (40×, N.A. ) 0.95, Zeiss, Plan Apochromat). To achieve better focusing, the laser beam was expanded 5× using a telescope lens arrangement. Movement on the z axis and large-scale movement on the x-y plane were carried out with a three-axis linear encoder stage (PI). The beam was controlled using a mechanical shutter (Uniblitz), whereas the beam intensity was adjusted by a motorized attenuator (Altechna). The stages, the shutter, and the attenuator were computer-controlled via a National Instruments LabVIEW program. A CCD camera was mounted behind a dichroic mirror for online monitoring of the polymerization process. This is possible because the refractive index of the copolymer changes during polymerization, and the illuminated structures become visible during the building process. The structures were fabricated in a layer-by-layer fashion with the last layer on the surface of the coverslip. After the completion of the component building process, the sample was developed for 1 h in 4-methyl-2-pentanone and rinsed with isopropanol. The samples were characterized by scanning electron microscopy. Before SEM analysis, they were coated with a 10-nm-thick palladium layer. Cytotoxicity Tests. For the cytotoxicity texts, murine fibroblast cell line NIH-3T3 was obtained from the American Type Culture Collection (Rockville, MD). The cells were suspended at a concentration of 105 cells/mL in Dulbecco’s modified Eagle’s medium (30) Serbin, J.; Ovsianikov, A.; Chichkov, B. Opt. Express 2004, 12(21), 5221– 5228. (31) Kawata, S.; Sun, H. B.; Tanaka, T.; Takada, K. Nature 2001, 412(6848), 697–698. (32) Meisel, D. C.; Deubel, M.; Hermatschweiler, M.; Busch, K.; Koch, W.; von Freymann, G.; Blanco, A.; Enkrich, C.; Wegener, M. Three-Dimensional Photonic Crystals. In Functional Nanomaterials for Optoelectronics and Other Applications; Lojkowski, W., Blizzard, J. R., Eds.; Trans Tech Publications: Zurich-Vetikon, Switzerland, 2003; Vol. 99-100, pp 55-64. (33) Serbin, J.; Egbert, A.; Ostendorf, A.; Chichkov, B. N.; Houbertz, R.; Domann, G.; Schulz, J.; Cronauer, C.; Frohlich, L.; Popall, M. Opt. Lett. 2003, 28(5), 301–303. (34) Farsari, M.; Filippidis, G.; Fotakis, C. Opt. Lett. 2005, 30(23), 3180– 3182.

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Figure 5. Cells spreading on glass surface after 2 days (a) and 7 days (c), and on polymer films after 2 days (b) and 7 days (d). The cells were cultivated for the respective days, submitted to staining, and observed with a fluorescence microscope. Live cells were stained only by the cell-permeable Live-Dye, fluorescing green. Dead cells can be stained by both the cell-permeable Live-Dye and the cell nonpermeable PI (red), resulting in the overlay of green and red which appears to be yellow-red.

(DMEM) with 10% fetal calf serum (FCS) and 1% gentamycin solution (GIBCO, Invitrogen, Karlsruhe, Germany) A cell suspension (1 mL) was added to the modified glass coverslips in a 24-well plate and cultured at 37 °C in 5% CO2 for 24 h. Before seeding the cells on the polymer surfaces, cells were grown to confluency, detached with 0.05% trypsin/EDTA (GIBCO, Invitrogen, Karlsruhe, Germany), and diluted in complete medium at an appropriate density. Cell growth and viability were assessed with a live-dead cell staining kit (BioVision, CA).

Acknowledgment. F.C. was supported by an EPSRC Life Science Interface Fellowship (Grant No. EP/C532066/1). The work of A.G. was supported by the EU Marie Curie Fellowship Program: ATLAS (MEST-CT-2004-008048). The work of C.R.

and B.N.C. was supported by the Marie Curie Transfer of Knowledge project NOLIMBA (MTKD-CT-2005-029194). A.O. and B.N.C. acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG), Project Transregio 37 and Rebirth Excellence Cluster. The work of D.S.A. was supported by PENED 2003 programme 03E∆581. Travel between Crete, Greece, and Hannover, Germany, was supported by an IKYDA/ DAAD travel grant. We are grateful to Mrs. Aleka Manousaki for expert technical assistance with SEM.

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