Large Scale Laser TwoPhoton Polymerization ...

Large Scale Laser TwoPhoton Polymerization Structuring for Fabrication of Artificial Polymeric Scaffolds for Regenerative Medicine M. Malinauskas, V. Purlys, A. Žukauskas, M. Rutkauskas, P. Danilevičius et al. Citation: AIP Conf. Proc. 1288, 12 (2010); doi: 10.1063/1.3521344 View online: http://dx.doi.org/10.1063/1.3521344 View Table of Contents: http://proceedings.aip.org/dbt/dbt.jsp?KEY=APCPCS&Volume=1288&Issue=1 Published by the American Institute of Physics.

Related Articles Confinement and viscoelastic effects on chain closure dynamics J. Chem. Phys. 136, 234903 (2012) Memory effects during the unbiased translocation of a polymer through a nanopore J. Chem. Phys. 136, 154903 (2012) Slow growth of the Rayleigh-Plateau instability in aqueous two phase systems Biomicrofluidics 6, 022007 (2012) Tunable spatial heterogeneity in structure and composition within aqueous microfluidic droplets Biomicrofluidics 6, 022005 (2012) Polymerization of actin filaments coupled with adenosine triphosphate hydrolysis: Brownian dynamics and theoretical analysis JCP: BioChem. Phys. 5, 09B604 (2011)

Additional information on AIP Conf. Proc. Journal Homepage: http://proceedings.aip.org/ Journal Information: http://proceedings.aip.org/about/about_the_proceedings Top downloads: http://proceedings.aip.org/dbt/most_downloaded.jsp?KEY=APCPCS Information for Authors: http://proceedings.aip.org/authors/information_for_authors

Downloaded 28 Nov 2012 to 78.60.203.55. Redistribution subject to AIP license or copyright; see http://proceedings.aip.org/about/rights_permissions

Large Scale Laser Two-Photon Polymerization Structuring for Fabrication of Artificial Polymeric Scaffolds for Regenerative Medicine M. Malinauskasa*, V. Purlysa, A. Žukauskasa, M. Rutkauskasa, P. Danilevičiusa, D. Paipulasa, G. Bičkauskaitėa, L. Bukelskisb, D. Baltriukienėb, R. Širmenisc, A. Gaidukevičiūtėd,V. Bukelskienėb, R. Gadonasa, V. Sirvydise and A. Piskarskasa a

Vilnius University, Physics Faculty, Department of Quantum Electronics, Laser Research Center, Saulėtekio ave. 10, LT-10223 Vilnius, Lithuania b Institute of Biochemistry, Vivarium, Mokslininkų str. 12, LT-08662 Vilnius, Lithuania c Vilnius University Hospital Santariškių Klinikos, Santariškių g. 2, LT-08661 Vilnius, Lithuania d Institute of Electronic Structure and Laser, Foundation for Research and Technology Hellas, Vassilika Vouton, 711 10 Heraklion, Crete, Greece e Vilnius University, Faculty of Medicine, Heart Surgery Center, Santariškių 2, LT-08661, Vilnius, Lithuania

Abstract. We present a femtosecond Laser Two-Photon Polymerization (LTPP) system of large scale three-dimensional structuring for applications in tissue engineering. The direct laser writing system enables fabrication of artificial polymeric scaffolds over a large area (up to cm in lateral size) with sub-micrometer resolution which could find practical applications in biomedicine and surgery. Yb:KGW femtosecond laser oscillator (Pharos, Light Conversion. Co. Ltd.) is used as an irradiation source (75 fs, 515 nm (frequency doubled), 80 MHz). The sample is mounted on wide range linear motor driven stages having 10 nm sample positioning resolution (XY - ALS130-100, Z - ALS130-50, Aerotech, Inc.). These stages guarantee an overall travelling range of 100 mm into X and Y directions and 50 mm in Z direction and support the linear scanning speed up to 300 mm/s. By moving the sample three-dimensionally the position of laser focus in the photopolymer is changed and one is able to write complex 3D (three-dimensional) structures. An illumination system and CMOS camera enables online process monitoring. Control of all equipment is automated via custom made computer software “3D-Poli” specially designed for LTPP applications. Structures can be imported from computer aided design STereoLihography (stl) files or programmed directly. It can be used for rapid LTPP structuring in various photopolymers (SZ2080, AKRE19, PEG-DA-258) which are known to be suitable for bio-applications. Microstructured scaffolds can be produced on different substrates like glass, plastic and metal. In this paper, we present microfabricated polymeric scaffolds over a large area and growing of adult rabbit myogenic stem cells on them. Obtained results show the polymeric scaffolds to be applicable for cell growth practice. It exhibit potential to use it for artificial pericardium in the experimental model in the future. Keywords: femtosecond laser microfabrication, photopolymerization, 3D nanolithography, artificial scaffolds, stem cells, regenerative medicine. PACS: 42.55.Xi, 87.15.rp, 87.17.Uv

INTRODUCTION Stem cell biology, science of materials and nanotechnology, are already yielding products to help sick and injured people. Tissue engineering is believed to be the technique to provide a solution of many health problems. Reconstruction of numerous tissues and organs such as skin, blood vessels, intestine, trachea, urological organs, pericardium and others are among them [1-5] Nowadays, multiform polymeric matrices or segments for primary autologous stem cell growing are being designed which can serve as a template for artificial tissue fabrication [6]. Tissue engineering principles focus on: (a) healthy cells, which have to be nonimmunogenic, easily isolated and highly responsive to distinct environmental cues, (b) suitable carriers for the in vitro cell differentiation and subsequent transplantation, and (c) a set of defined bioactive molecules driving the process of differentiation and maturation [7]. Therefore, autologous stem cell growing is appropriate technology for the fabrication of artificial tissue, but demand in development of technologies for 3D cell culture scaffolds is still required [8]. Existing techniques used up to day lack of structuring resolution, are restricted in true 3D geometry or limited to the processible materials and relatively low CREDIT LINE (BELOW) TO BE INSERTED ON THE FIRST PAGE OF EACH PAPER CP1288, Emerging Trends and Novel Materials in Photonics, International Commission for Optics Topical Meeting edited by N. A. Vainos, S. Pissadakis, S. Couris, E. Paspalakis, and I. Koutselas © 2010 American Institute of Physics 978-0-7354-0843-2/10/$30.00

12


throughput. On the other hand, finite understanding of the interaction of cells with substrates results in high demand of new technologies enabling fabrication of 3D scaffolds for testing the biocompatibility of 3D texture. Novel technologies emerge that seem to meet the requirements for 3D scaffolds fabrication [9]. Direct laser writing is a very promising approach to optical maskless lithography, which offers flexibility, ease of use, and cost effectiveness in R&D processing [10, 11]. This equipment offers rapid patterning at sub-micron resolutions, compromising between performance and costs when working with feature sizes of approximately 200 nm or greater comparing to µ-stereolithography or electron beam writing [12–15]. Compared to alternative technologies it provides unique ability of 3D sculpting of any geometry features [16, 17]. LTPP is also attractive for easily scalability and flexibility of structuring arbitrary textures in various biocompatible polymers [18, 19]. However, for application in tissue engineering much effort still has to be applied both in improving fabrication technology of scaffolds and understanding cell behaviour at micro-scale [20]. In this paper we present the results of progress in fabrication of 3D scaffolds applying constructed LTPP system and testing their biocompatibility using rabbit stem cells.

PHOTOSENSITIVE MATERIALS In our experiment we have used three different negative photoresins: custom made acrylate based material AKRE19 [11, 19], hybrid organic-inorganic and SZ2080 (ORganicaly MOdified SILica SZ2080, FORTH) [21, 22] and biodegradable PEG-DA-258 (PolyEthylenGlycol Di-Acrylate of M.w. = 258, Sigma-Aldrich GmbH) [23, 24]. The prepolymers were photosensitized adding 1 - 2 wt.% of thioxanthen-9-one or 2-Benzyl-2-dimethylamino-1-(4morpholinophenyl)-butanone-1 photoinitiators (Sigma-Aldrich GmbH). Chemical formulas and developers of the used materials are given in table 1. TABLE 1. Photosensitive pre-polymer materials.

Material

Chemical formula

Developer

AKRE19

C18H21N3O9

ethyl alcohol

SZ2080

C4H12SiZrO2

methyl isobutyl ketone

PEG-DA-258

C8H10O4

water

Samples for fabrication were prepared by drop-casting the photopolymer on a cover glass substrate. After laser processing samples were treated with the appropriate organic solvent in order to wash out unexposed material. Light exposed photopolymer undergoes polymerization and becomes insoluble in the developer, polymerized features sustain during development process. In this way, the free-standing structures on a glass substrate were obtained. Scanning Electron Microscopy (SEM) was applied to investigate the microstructured scaffolds.

FEMTOSECOND LASER TWO-PHOTON POLYMERIZATION SYSTEM In our experiments LTPP system was constructed and tested for microstructuring of photopolymers in 3D over a large area [11, 19]. A schematic illustration of the fabrication system is shown in Fig. 1. Yb:KGW femtosecond laser oscillator (Pharos, Light Conversion. Co. Ltd.) is used as an irradiation source. The laser parameters are: repetition rate – 80 MHz, average power – 1.2 W, 75 fs pulse duration, 1030 nm and 515 nm (second harmonic) central wavelengths. The expanded femtosecond laser beam is guided through the objective, focusing femtosecond laser beam into the volume of the photoresin. The sample is mounted on XYZ wide range positioning stages. The positioning system consists of linear motor driven stages (Aerotech, Inc.): XY - ALS130- 100, Z – ALS130-50. These stages ensure an overall travelling range of 100 mm into X and Y directions and 50 mm in Z direction and support the scanning speed up to 300 mm/s. Upon irradiation the pre-polymer undergoes transition from liquid to solid (or from gel to solid) and results to the change in refractive index. It enables wide-field transmission microscopy to be used for monitoring the manufacturing process in real time. A microscope is built by assembling its main components: a source of red light provided by LED, a CMOS (mvBlueFOX-M102G, Matrix Vision GmbH) camera and a video screen. The ability to image photostructuring while performing LTPP is an important feature for successful fabrication process. It is of utmost to anchor the microstructures to the substrates for them to survive the washing step of the unsolidified resin. Control of

13


all equipment is automated via custom made computer software “3D-Poli” specially designed for LTPP applications. By moving the sample three-dimensionally inside the resin the position of laser focus is changed and one is able to write complex 3D structures. Structures can be imported from Computer Aided Design (CAD) files or programmed directly. This LTPP system was tested for structuring in various photosensitive materials at large scale [11]. The ability to scale up and speed up the fabrication is ensured by changing laser beam focusing objectives in the range from 100x NA = 1.4 to 8x NA = 0.2, thus at the sacrifice of the resolution from 150 nm to 20 µm [19].

FIGURE 1. LTPP fabrication setup. Femtosecond beam is guided to nonlinear crystal (NC), reflected by dichroic mirror (DM) and coupled to objective lens (OL). Sample is fixed on XYZ stages which are computer controlled (PC). LED provides illumination needed for CMOS camera to monitor (OM) the fabrication process online.

FABRICATION OF 2D AND 3D SCAFFOLDS Various in form and dimensions scaffolds have been successfully produced and shown in Fig. 2. Throughput is an important factor for the technology to be widely applied. Fabrication duration time t of specific scaffold can be estimated by formula 1:

t=

xyzF . Rv

(1)

Here x, y and z is the dimensions of the structure, F - filling factor, R - resolution and v is sample scanning speed. It is seen that by designing the CAD model increase of the structure size affects the fabrication time dramatically, though appropriately chosen R and F can reduce it, while scanning speed v is a user set parameter and is limited only by sample positioning stages. For example, it took up to 60 h to fabricate 3D woodpile structure having dimensions of 5x5x0.02 mm3 at 1000 µm/s sample scanning speed (Fig 2 a). In comparison, only 1h is needed to produce 2D grid having lateral dimensions of 10x10x0.05 mm3 at scanning speed 500 µm/s (Fig. 2 b). Though the fill factor F of both, woodpile structure and 2D grid was comparable, the resolution R was 500 nm and 20 µm, respectively.

14


FIGURE 2. Optical (a), b)) and SEM (c), d)), pictures of microstructured artificial polymeric scaffolds: a) 3D woodpile structure of SZ2080 on glass substrate having lateral resolution d = 500 nm and axial z = 1500 nm, b) 2D grid of PEGDA-258 on stainless steel substrate having 20 µm linewidth, c) honey comb structure of SZ2080, d) woodpile structure of PEG-DA-258.

STEM CELL GROWING ON THE POLYMERIC SCAFFOLDS Adult stem cells derived from rabbit muscle were used for cell-based artificial tissue fabrication on the LTPP engineered scaffolds. Myogenic stem cells were isolated from skeletal muscle tissue and grown in Iscove’s modified Dulbecco minimum essential medium (Sigma-Aldrich GmbH) supplemented with 10% fetal calf serum (Sigma-Aldrich GmbH) in multiple polystyrene tissue culture plates (Orange Scientific, Switzerland) [25, 26]. A qualitative analysis of the biocompatibility of polymers was assessed according adult stem cell viability. The cells were maintained on the non-structured polymeric film for 48 h and their viability was registered by staining with dyemix solution of 100 μg/ml acridine orange (Molecular probes Inc.) and 100 μg/ml ethidium bromide (Sigma-Aldrich GmbH). A thin film of dye mixture prepared in culture medium (5:95) was added on the cell monolayer grown on the polymeric surface. Acridine orange is taken up by viable cells. It intercalates into double-stranded DNA and makes it appear green. Etidium bromide is only taken up by nonviable cells, it intercalates into DNA, making it appear orange [27]. Cell visualization was accomplished and photomicrographs were taken by using fluorescent microscope (Nikon Eclipse TS100/100-F) equipped with a CCD camera (Lumenara Infinity 2C). After, primary adult myogenic stem cell line was used for the constructing of cell-based artificial tissue on the LTPP engineered scaffolds. Prior the cell inoculation to well containing the scaffold, they were stained with 10 µg/ml diamidino-2-phenylindole (DAPI). DAPI is the classic blue fluorescent probe for viable cells that fluoresces brightly when it is bound to DNA. Therefore, DAPI stained myogenic stem cells were cultured on the AKRE19 material or hybrid organic-inorganic, SZ2080 and PEG-DA-258 LTPP engineered scaffolds. Cells were inoculated at a density of 60000 cells per ml, after 72 h cultivation the proliferation of stem cells on the LTPP scaffolds was assessed microscopically.

15


RESULTS For modern implants and cell-based artificial tissues, it is important to engineer structures with characteristics that closely emulate nature. The advances in various fields of science enable and enforce the scientists to search for novel biocompatible biomaterials with distinct properties and innovative design which are necessary for engineering contemporary composite tissue with a purposeful orientation towards anatomic structures. Following the data we conclude that three our tested polymers were biocompatible because the cells grown on them appeared green after staining with AO&EB (Fig. 3 A).

A

PEG-DA-258

SZ2080

AKRE19

PEG-DA-258

SZ2080

AKRE19

B

FIGURE 3. A – alive (AO-stained) stem cells grown on the non-structured various polymeric surfaces: PEG-DA-258, SZ2080 and AKRE19; B – DAPI-stained stem cells grown on the LTPP fabricated three-dimensional PEG-DA-258, SZ2080 and AKRE19- derived microstructured scaffolds. Our LTPP fabricated 3D microstructured polymers distinguish by stable internal architecture as well as flexibility and enables the constructive be suitable for biomedical applications (Fig. 3 B). Adult stem cells herewith LTPP nanostructured polymers represent an important building materials for regenerative medicine and tissue engineering.

CONCLUSIONS We have constructed LTPP system based on Yb:KGW frequency doubled femtosecond laser in combination with linear motor-driven stages for routine high resolution and throughput 3D micro- and nano-fabrication. It was used for producing artificial polymeric scaffolds applied for stem cell growth. Polymeric scaffolds of biocompatible photopolymers up to cm in lateral size were successfully fabricated having sub-micrometer spatial resolution. Adult rabbit myogenic stem cells proliferation tests show artificial scaffolds to be applicable for biomedicine practice. However, further efforts, like investigation of scaffolds biocompatibility and biodegradability in vivo as well as structures mechanical properties have to be done before the regenerative medicine accepts it into practice. It could be potential to use it for artificial pericardium in the experimental model of laboratory animals in near future.

ACKNOWLEDGMENTS This work is supported by the Lithuanian State Science and Studies Foundation Grants B09/08 (Laser MicroProcessing with High Repetition Femtosecond Pulses - Femtoprocessing) and B-07041 (Development of the models to regenerate heart structures employing stem and specialized cells also biological tissues). We gratefully acknowledge M. Farsari (Foundation for Research and Technology Hellas, [email protected]) for providing zirconium containing sol-gel hybrid photosensitive material SZ2080. M. Malinauskas thanks project NOLIMBA for covering the travelling expenses.

16


REFERENCES 1. A. Charruyer and R. Ghadially, Stem cells and tissue-engineered skin. Skin Pharmacol. Physiol. 22 (2), 55–62 (2009). 2. W.H. Zimmermann, I. Melnychenko and T. Eschenhagen, Engineered heart tissue for regeneration of diseased hearts. Biomaterials. 25 (9),1639–1647 (2004). 3. A. Mantesso and P. Sharpe, Dental stem cells for tooth regeneration and repair. Expert Opin Biol Ther. 9 (9), 1143–1154 (2009). 4. A. Subramanian, U.M. Krishnan and S. Sethuraman, Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration. J. Biomed. Sci. 16, 108 doi:10.1186/1423-0127-16-108 (2009). 5. A. Atala, Tissue engineering, stem cells, and cloning for the regeneration of urologic organs Clin. Plast. Surg. 30 (4), 649–667 (2002). 6. J. Ringe, C. Kaps, G.-R. Burmester and M. Sittinger, Stem cells for regenerative medicine: advances in the engineering of tissues and organs, Naturwissenschaften 89, 338–351, (2002). 7. M. Sittinger, J. Bujia, N. Rotter, D. Reitzel, W.W. Minuth and G.R. Burmester, Tissue engineering and autologous transplant formation: practical approaches with resorbable and new cell culture techniques. Biomaterials 17, 237–242 (1996). 8. T. Hodgkinson, X.F. Yuan and A. Bayat, Adult stem cells in tissue engineering. Expert Rev. Med. Devices. 6 (6), 621–640 (2009). 9. A. Ovsianikov, S. Schlie, A. Ngezahayo, A. Haverich and B.N. Chichkov, Two-photon polymerization technique for microfabrication of CAD-designed 3D scaffolds from commercially available photosensitive materials, J. Tissue Engin. Regen. Med. 1, 443-449 (2008). 10. F. Claeyssens, E.A. Hasan, A. Gaidukevičiūtė, D.S. Achilleos, A. Ranella, C. Reinhardt, A. Ovsianikov, X. Shizhou, C. Fotakis, M. Vamvakaki, B.N. Chichkov and M. Farsari, Three-dimensional biodegradable structures fabricated by two-photon polymerization, Langmuir, 25 (5), 3219–3223 (2009). 11. M. Malinauskas, V. Purlys, M. Rutkauskas and R. Gadonas, Two-photon polymerization for fabrication of three-dimensional micro- and nanostructures over a large area, SPIE Proc. 7204, 72040C (2009). 12. S.D. Gittard, R.J. Narayan, J. Lusk, P. Morel, F. Stockmans, M. Ramsey, C. Laverde, J. Phillips, N.A. Monteiro-Riviere, A. Ovsianikov and B.N. Chichkov, Rapid prototyping of scaphoid and lunate bones, Biotechnol. J. 4, 129–134 (2009). 13. J. Stampfl, S. Baudis, C. Heller, R. Liska, A. Neumeister, R. Kling, A. Ostendorf and M. Spitzbart, Photopolymers with tunable mechanical properties processed by laser-based high-resolution stereolithography, J. Micromech. Microeng. 18, 125014 doi: 10.1088/0960-1317/18/12/125014 (2008). 14. D.R.S. Cumming, S. Thoms, S.P. Beaumont and J.M.R. Weaver, Fabrication of 3 nm wires using 100 keV electron beam lithography and poly(methyl methacrylate) resist, Appl. Phys. Lett. 68 (3), 322–324 (1996). 15. W. Chen and H. Ahmed, Fabrication of 5-7 nm wide etched lines in silicon using 100 keV electron-beam lithography and polymethylmethacrylate resist, Appl. Phys. Lett. 62 (13), 1499–1501 (1993). 16. C. Schizas, V. Melissinaki, A.Gaidukevičiūtė, C. Reinhardt, C. Ohrt, V. Dedoussis, B.N. Chichkov, C. Fotakis, M. Farsari and D. Karalekas, On the design and fabrication by two-photon polymerization of a readily assembled micro-valve, Int. J. Adv. Manuf. Technol. 0 (0), 1–7 (2009). 17. M. Malinauskas, P. Danilevičius, D. Baltriukienė, M. Rutkauskas, A. Žukauskas, Ž. Kairytė, G. Bičkauskaitė, V. Purlys, D. Paipulas, V. Bukelskienė and R. Gadonas, 3D artificial polymeric scaffolds for regenerative medicine fabricated by femtosecond laser, Lithuanian J. Phys. (submitted 2009). 18. S. Passinger, A. Ovsianikov, R. Kiyan, C. Reinhardt, A. Ostendorf and B. Chichkov, Two-Photon Polymerization for industrial applications, LPM Proc. (2008). 19. M. Malinauskas, V. Purlys, M. Rutkauskas, A. Gaidukevičūtė and R. Gadonas, Femtosecond Visible Light Induced Two-Photon Photopolymerization for 3D Nanostructuring of Various Polymeric Materials, Lithuanian J. Phys. (submitted 2009). 20. A. Gaidukevičiūtė (private communication). 21. A. Ovsianikov, J. Viertl, B. Chichkov, M. Oubaha, B. MacCraith, I. Sakellari, A. Giakoumaki, D. Gray, M. Vamvakaki, M. Farsari and C. Fotakis, Ultra-Low Shrinkage Hybrid Photosensitive Material for Two-Photon Polymerization Microfabrication, ACS Nano, 2 (11), 2257–2262 (2008). 22. A. Ovsianikov, A. Gaidukevičiūtė, B.N. Chichkov, M. Oubaha M, B.D. MacCraith, I. Sakellari, A. Giakoumaki, D. Gray, M. Vamvakaki, M. Farsari and C. Fotakis, Two-photon polymerization of hybrid Sol-Gel materials for photonics applications, Laser Chemistry 493059, 1–7 (2008). 23. A. Ovsianikov, A. Ostendorf and B. Chichkov, Three-dimensional photofabrication with femtosecond lasers for applications in photonics and biomedicine, Appl. Surf. Sci. 253, 6599–6602 (2007). 24. A. Ovsianikov, M. Malinauskas, S. Schlie, A. Ngezahayo and B. Chichkov, Two-photon polymerization of Poly(Ethylene Glycol) materials for biomedical applications, (in preparation 2009). 25. V. Bukelskienė, D. Baltriukienė, D. Bironaitė, A. Imbrasaite, R. Širmenis, M. Balčiūnas, E. Žurauskas and A. Kalvelytė, Musclederived primary stem cell lines for heart repair, Sem. Cargiol. 11, 99–105 (2005). 26. R. Širmenis, V. Bukelskienė, V. Domkus and V. Sirvydis, Cellular cardiomyoplasty: isolation and cultivation of skeletal muscle satellite cells, Acta Med. Lituanica. 6, 178–181 (1999). 27. S. Mercille and B. Massie, Induction of apoptosis in nutrient-deprived cultures of hybridoma and myelonoma cells, Biotechnol. Bioeng. 44, 1140–1154 (1994).

17
