Self-standing aligned fiber scaffold fabrication by two photon photopolymerization
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Development of materials and fabrication techniques lead the growth of three-dimensional cell culture matrices in biomedical engineering. In this work, we present a method for fabricating self-standing fiber scaffolds by two-photon polymerization induced by a femtosecond laser. The aligned fibers are 330 μm long with a diameter of 6–9 μm. Depending on the pitch of the aligned fibers, various cell morphologies are distinguished via three-dimensional images. Furthermore, the morphologies of fibroblast cells (NIH-3T3) and epithelial cells (MDCK) on the fiber scaffolds are studied to show the effect of high curvature (3–4.5 μm radii) on cell morphology. NIH-3T3 cells that contain straight pattern of actin microfilament bundles are extended and partly wrap single fibers or tend to reside between fibers. On the other hand, MDCK cells that contain circular pattern of actin microfilament bundles cover the fiber peripheral surface exhibiting high aspect ratio elongation. These results indicate that cell morphology on fiber scaffolds is influenced by the pattern of actin microfilament bundles.
KeywordsCell morphology Fiber scaffold Laser manufacturing Two-photon polymerization Fibroblast cell Epithelial cell
Almost every cell in human body inhabits three-dimensional (3D) tissues that cannot be accurately represented by conventional two-dimensional (2D) culturing surfaces. In order to engineer virtually human tissue, artificial 3D cell culture scaffolds such as 3D fibrous matrices have been designed and applied to support cell and tissue growth (Hutmacher 2001; Lee et al. 2008). The scaffolds are typically fabricated with fiber based structures by fiber bonding (Mikos et al. 1993), electrospinning (Chen et al. 2007; Ji et al. 2006; Murugan and Ramakrishna 2007), weaving (Chen et al. 2004; Cooper et al. 2005), fused-deposition modeling (Yeong et al. 2004) and carbon nanotube yarns (Galvan-Garcia et al. 2007). Fibrous structures tend to enhance cell adhesion, proliferation and differentiation functions (Lee et al. 2008). Just a few studies explored cell behavior on a single fiber or the influence of the dimensions of aligned multiple fibers on cell and tissue growth, even though groove width seems to play an important role in the control of cells on patterned surfaces (Curtis and Wilkinson 1997), largely due to lack of self-standing, position controllable fiber fabrication techniques.
In addition to developing 3D cell culture scaffolds, it is crucial to understand the interaction of biological cells with cell culture substrates for development of tissue engineering. Cell behavior, such as cell adhesion, proliferation and gene expression, can be directed by the substrate geometry and surface chemistry (Clark et al. 1990; Curtis and Wilkinson 1997; den Braber et al. 1995; Dunn and Heath 1976; Kaiser et al. 2006; Meyle et al. 1994; Rebollar et al. 2008; Rovensky et al. 1999; Rovensky and Samoilov 1994; Suh et al. 2004; Thomas et al. 1999, 2002). Various studies on these effects have been reported, most utilizing substrates bearing nano-scale (Rebollar et al. 2008) to micro-scale (Clark et al. 1990; Curtis and Wilkinson 1997; den Braber et al. 1995; Kaiser et al. 2006; Meyle et al. 1994) patterned surfaces as well as chemically defined surfaces (Suh et al. 2004; Thomas et al. 1999, 2002). In addition to the patterned flat surface, cylindrical substrates with high degree of curvature have been shown to affect morphogenetic response of cultured cells (Dunn and Heath 1976; Rovensky et al. 1999; Rovensky and Samoilov 1994).
In this study, two-photon induced polymerization by ultra-fast laser radiation has been used to generate self-standing, position controllable fiber scaffolds to study cell-material interactions. Compared with conventional stereo lithography, the two-photon polymerization process can be confined to cure the photocurable resins (PR) only near the laser focal volume (Doraiswamy et al. 2006; Drakakis et al. 2006; Kawata et al. 2001), hence enabling fabrication of arbitrary three-dimensional structures. The main objective of this work is to develop high resolution self-standing, position controllable fiber fabrication techniques and to demonstrate cell culture on the fibers. The high aspect ratio fiber fabrication via two-photon photopolymerization induced by high repetition femtosecond laser irradiation is based on the combined mechanisms of self-focusing (Kewitsch and Yariv 1996), self-growing (Shoji et al. 2002) and accumulation (Hidai et al. 2008). Two-photon polymerization techniques typically utilize scanning of the laser beam to create structures by stitching the tiny voxels cured within the focal zone. In a striking difference from these studies, our technique enables fabrication of high aspect ratio (∼180:1) fibers with reasonable throughput as scanning is avoided (Hidai et al. 2008). Biological effects on cell shape are demonstrated using fibroblast cell lines (NIH-3T3) and epithelial cell lines (MDCK). We examine various 3D morphologies of cells cultured on fiber scaffolds and report their dependence on the cell type.
2 Materials and methods
2.1 Photocurable resin
The PR used for the fiber growth was a UV curable organic–inorganic hybrid polymer (ORMOCER®, US-S4, Micro resist technology). ORMOCER® is non-toxic, biologically inert and optically transparent over the 400–1,600 nm wavelength range (Doraiswamy et al. 2006).
After the hard bake, uncured PR was filled between the glass plates and fibers were fabricated by high-repetition-rate femtosecond laser irradiation (Fig. 1(b)). A femtosecond laser beam (pulse width: <500 fs, repetition rate: 1 MHz, wavelength: 1,045 nm, typical M2: 1.3, FCPA μJewel D-400, IMRA America, inc.) was frequency-doubled to the wavelength of ∼523 nm and focused at ∼500 μm below the bottom glass plate/PR interface by illuminating from the top through a ×5 microscope objective (M plan apo, N.A. = 0.14, Mitutoyo). The power of the laser beam emitted downstream of the objective lens was measured by a power meter and controlled by a half wave plate and a polarizing beam splitter, and was set at ∼3 mW. The exposure duration was controlled by a mechanical shutter and set at 0.2 s. The sample was placed on a motorized X–Y stage. Fibers were fabricated at desired positions by the shutter and stages controlled by a PC.
After the laser irradiation (Fig. 1(c)), the samples were baked at 110°C for 10 min, then developed with ORMODEV® (Micro resist technology) for 30 min, rinsed with iso-propanol (IPA) three times, deionized (DI) water with 60 mg/mL asolectin (BioChemika), dipped in DI water and exposed with UV lamp for 30 min to cure completely. The sample was afterward dipped in 70% ethanol to sterilize, exposed for coating to 20 μg/mL fibronectin (FN) (Sigma) in phosphate buffered saline (PBS) (Gibco Invitrogen) for 1 h, and finally rinsed with PBS three times.
2.3 Scanning electron microscopy
Surface morphology and diameter of the fibers were characterized using a SEM (LEO 1550) at 5 kV acceleration voltage. The sample was dried out after washing with IPA and sputter coated with gold (30 nm thick) for SEM inspection.
2.4 Cell culture and imaging
Cell growth and viability studies were performed using a fibroblast cell line (NIH-3T3) and epithelial cell line (MDCK). The cells were grown in Dulbecco’s Modified Eagle Medium (Gibco Invitrogen), 10% fetal bovine serum (FBS) (Gibco Invitrogen) and 100 U/mL penicillin (Gibco Invitrogen). The cells were incubated in sterile polystyrene Petri dishes with the fiber scaffold as shown in Fig. 1(d), and stored in a 37°C and 5% CO2 culture incubator.
For time-resolved image acquisition, cells were cultured on fibers and maintained at 37°C in CO2 independent media (Gibco Invitrogen), 10% FBS, 100 U/mL penicillin, and 1% GlutaMAX (Gibco Invitrogen) on a microscope stage. Images were taken every 1 min with a digital CCD camera (Retiga 2000R cooled, Qimaging).
After incubation for 1 day, the cells were fixed with 3.7% formaldehyde (Fisher Scientific), and then permeabilized with 0.1% TritonX-100 (Fisher Scientific). Actin cytoskeleton and nucleus were stained with 330 nM alexa fluor 488 phalloidin (Invitrogen) for 40 min, and 300 nM DAPI (Invitrogen) for 4 min, respectively. Samples were kept in PBS and turned over for upright confocal microscope observation. A 510 Meta UV/VIS confocal microscope (Zeiss) with a ×63 N.A. = 1.0 W Plan Apochromat dipping objective lens (Zeiss) was utilized to visualize the morphology of fluorescent-stained cells on fibers. Z-scan pictures were obtained to visualize the cell morphology.
3 Results and discussion
3.1 Fiber fabrication
In-situ observation of the fiber growth showed that it commenced in the neighborhood of the geometrical focus and then self-propagated towards the light source without scanning of either the focusing lens or the sample (Hidai et al. 2008). While PR is cured only from the surface by conventional photolithography with UV laser, under femtosecond laser illumination the fiber can be cured at an arbitrary position inside the resin within the depth of focus due to two-photon absorption (Doraiswamy et al. 2006; Drakakis et al. 2006; Kawata et al. 2001).
We previously reported that fibers of ∼1.8 mm length could be readily fabricated after ∼1 s illumination with the ∼5 mW laser power when the polymer was filled in a thick glass cuvette. The fiber diameter was about 10 μm in the middle and decreased toward the edges (Hidai et al. 2008). Through process optimization, fibers of 0.33 mm in length were fabricated bridging the two glass plates.
3.2 Cell morphology on aligned fiber scaffolds
In order to demonstrate the biocompatibility of the fabricated fiber, fibroblast cell line (NIH-3T3) and epithelial cell line (MDCK) were cultured on the fibers coated with FN. The samples were dipped in culture solution, and then cells spread and attached on fibers.
We have developed a new fabrication technique to produce self-standing and controlled pitch fiber scaffolds by using two-photon polymerization effected by a high repetition rate ultra-fast laser. A cell-growing platform was chosen with the fiber diameter set at ∼6–9 μm and the length at ∼0.33 mm. Fibroblast cell lines (NIH-3T3) and epithelial cell lines (MDCK) were cultured on fibronectin coated fibers to demonstrate the biocompatibility of the fiber scaffolds and compare the cell morphologies depending on cell type and pitch of aligned fibers. This technique can be useful for fundamental level studies on the influence of microenvironment on cells and tissue engineering.
Support by the U.S. National Science Foundation under grant DMI-0556363 is gratefully acknowledged.
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