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Effects of mechanical stimuli and microfiber-based substrate on neurite outgrowth and guidance
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 101, No. 2, 120–126. 2006 DOI: 10.1263/jbb.101.120
© 2006, The Society for Biotechnology, Japan
Effects of Mechanical Stimuli and Microfiber-Based Substrate on Neurite Outgrowth and Guidance In Ae Kim,1 Su A Park,1 Young Jick Kim,2 Su-Hyang Kim,1 Ho Joon Shin,1 Yong Jae Lee,1 Sung Goo Kang,3 and Jung-Woog Shin1* Department of Biomedical Engineering, Inje University, 607 Eubang-Dong, Gimhae, Gyongnam, Republic of Korea,1 Department of Dental Laboratory Science, College of Health Sciences, Catholic University of Pusan, 9 Bugok 3-dong, Busan, Republic of Korea,2 and School of Biotechnology and Biomedical Science, Inje University, 607 Eubang-Dong, Gimhae, Gyongnam, Republic of Korea3 Received 1 September 2005/Accepted 29 October 2005
We introduced mechanical stimuli and micropatterned substrate with microfibers to investigate their effects on neurite outgrowth along with nerve growth factor in vitro. Two types of surface morphology were used: a surface that was coated by laminin alone and a surface where in microfibers was added on the laminin surface. PC-12 cells were seeded on both surface types and cultured for 2 d. The magnitudes of shear stresses ranged from 0.10 to 1.50 Pa. Two days after stimulation by shear stress, neurite outgrowth and its direction were measured by F-actin staining and digital image processing. When a shear stress of 0.50 Pa was applied, neurons were most highly aligned with microfibers. The average length of neurite outgrowth with microfibers was largest at a shear stress of 0.25 Pa. The results suggest that micropatterned fibers and fluid-induced shear stress are promising for stimulating neurite outgrowth in a desired direction. [Key words: neurite outgrowth length, microfiber-based substrate, PC-12 cells, fluid flow shear stress, electrospinning, nerve regeneration]
tor. When the voltage reaches a critical value, the charge overcomes the surface tension of the deformed drop of a suspended polymer solution formed on the tip of a syringe, and a jet is produced (7, 8). This is a straightforward, costeffective means of producing ultrafine fibers with diameters ranging from microns to a few nanometers. This method requires a small amount of polymer, such as poly(D,L-lactideco-glycolic acid) (PLGA), poly(glycolic acid) (PGA), silk, fibrinogen, collagen, or polymer composites (5, 8). In addition, the electrospun fibrous structure improves cell adhesion, proliferation, and ECM production (6). Another important factor on nerve regeneration is the mechanical environment. The mechanical environment plays a central role in the physiology of various tissues. Shear stress affects both mechanoreception, such as ion channels and integrins/focal adhesions, and response, such as intracellular calcium and nitric oxide production, and cytoskeletal remodeling (9). In particular, fluid-induced shear stress also induces the cellular orientation of osteoblasts, fibroblasts, and endothelial cells. Such stimulation also affects cellular migration and matrix outgrowth (10). Lee et al. (10) observed the effects of shear stress on cell orientation in canine arterial smooth muscle cells (SMCs) and quantified the time and magnitude dependences of shear stress alignment, and found that the alignment of SMCs induced at a high shear stress for longer periods. Klein-Nulend et al. (11) reported that a pulsatile fluid shear stress stimulates osteoblasts and osteocytes differently; when osteoblasts are subjected to a pulsatile fluid flow for 24 h, the cells become
Axons can regenerate spontaneously over relatively short distances (~5 mm), but not over large gaps in the adult peripheral nervous system (PNS). Although regeneration occurs after injury, it does not always result in functional recovery, because regenerating axons are usually directed toward inappropriate targets (1). Therefore, many studies have shown the effects of extracellular matrixes, microgrooved surfaces, new biologically compatible scaffolds, collagen, and fibronectin coating on the growth direction of axons (2–5). In many cell types including neurons, cell survival and growth are influenced by a cell extracellular matrix (ECM) signal. Therefore, various scaffolds based on ECM components, such as laminin or 3D-collagen gels, have been studied (3). However, the directing effects of these structures are very limited. One method that has been attempted is the micropatterning of substrate surfaces. Most micropatterned substrates have been fabricated in the form of microgrooves using ion beams, lasers, or microstamps on which laminin are adsorbed. In addition, it has been reported that neurite guidance and outgrowth are affected by the shapes, dimensions, and raw materials of micropatterned substrates (2–5). Recently, electrospinning has been utilized to produce patterned structures (6). Electrospinning is a fiber manufacturing method that utilizes a high electrical voltage. A high electric field is generated between a polymer fluid contained in a syringe with a capillary tip and a metallic collec* Corresponding author. e-mail: [email protected] phone: +82-55-320-3317 fax: +82-55-327-3292 120
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longer and prostaglandin E2 (PGE2) production increases. However, the effects of shear stress on nerve cells have been rarely reported. Recently, the effects of shear stress on Schwann cells in neurite outgrowth have been investigated. Chafik et al. (12) reported that shear stress is an important component of the natural environment for axon regeneration and that shear stress enhances cellular adhesion, proliferation, and alignment of Schwann cells. In this study, we fabricated microfibers (~3–5 µm in diameter) and deposited them on a substrate parallel to the direction of shear stress. The specific aim of using electrospun micron-sized fibers is to designate a guide for the neurite outgrowth under mechanical stimulation, that is, shear stress compared with other substrates without fibers. The fibers were made smaller than 5 µm considering the cell size and the diameter of neurite. In addition, the small diameter (<5 µm) of the fiber was expected not to disturb the shear stress distribution considering the height of the flow chamber as shown later (Fig. 2). On the basis of the rationale described above, we investigated the effects of shear stress on the neurite outgrowth of PC-12 cells along with changes in the surface morphology of the substrate.
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FIG. 1. Schematic diagram of fluid flow system. The flow system consisted of four consecutive chambers, two reservoirs, and a roller pump.
MATERIALS AND METHODS Preparation of cells and substrate Neurons are more fastidious in their choice of substrate than most other cells. Therefore, a laminin coating was applied for the adhesion of PC-12 cells. Coverglasses (24×36 mm) were coated with a 0.1% laminin solution (0.2 µg/cm2; Sigma, St. Louis, MO, USA) overnight in a petridish, and then rinsed twice with Hank’s Balanced Salt Solution (HBSS). Microfiber-patterned substrates were fabricated by electrospinning. The bulk copolymer of 50 :50 poly(D,L-lactide-co-glycolic acid) (PLGA; Alkermes, Cincinati, OH, USA) was dissolved in a solvent at 40°C to obtain a 50 wt% solution. The solvent consisted of a 1: 1 mixture of N,N-dimethylformamide (DMF; Junsei Chemical, Tokyo) and tetrahydrofuran (THF; Junsei Chemical). For electrospinning, the polymer solution was placed in a 5-ml plastic syringe fitted to an 18-gauge needle. To deposit the aligned microfibers on the laminin surface, a laminin-coated coverglass was glued to a rotating collecting target. To spray the solution, 10 kV was applied between the needle and the collecting target using a high-voltage power supply. The needle was 6.5 cm from the rotating target. The collecting target had a surface velocity of 4.2 m/s. A step motor was used for accurate speed control and the control software used was Turbo C (ver. 3.0; Boland International, Cupertino, CA, USA). Rat pheochromocytoma cells (PC-12) were obtained from the Korean Cell Line Bank (KCLB, accession no. 21721). The cells were cultured in Dulbecco’s modified Eagle medium high-glucose (DMEM-HG), containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT USA), 100 U/ml penicillin (Sigma, St. Louis, MO, USA), and 100 mg/ml streptomycin (Sigma) in an incubator at 37°C with 5% CO2. Design of fluid flow system Figure 1 shows the fluid flow system used for applying shear stress. There are four chambers connected in series, two reservoirs, and a roller pump (Masterflex; Cole-Parmer Instrument Company, Vernon Hills, IL, USA) in the system. One of the reservoirs was open to the atmosphere and the culture medium in the flow circuit was equilibrated with 95% air and 5% CO2 (13). The flow chambers included a flow section 1
FIG. 2. Flow chamber used for applying shear stress: (A) General picture of chamber with cover plate. (B) Schematics of chamber. The flow chamber included a flow section 1 mm in height and 30 mm in width.
mm in height (h) and 30 mm in width (b) (Fig. 2). The pump was controlled using LabVIEW (ver. 6.1i; National Instruments, Austin, TX, USA) using a personal computer. Under the assumption of a Newtonian fluid and fully developed laminar flow, the shear stress applied to the cells was calculated using τ = 6µQ/h2b where µ is dynamic viscosity, Q is flow rate, h is height and b is width. For the calculation, 9.6 ×10–4 Pa⋅s was used as the viscosity (µ) of the culture medium (13). Using computational fluid dynamics (CFD) analysis, we simulated the flow pattern in the fluid chamber. A two-dimensional finite element model was constructed. Using this model, the distri-
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TABLE 1. Classification of experimental groups according to pattern and shear stress magnitude Group L (only laminin-coated) LC (LS at 0 Pa): control LS at 0.10 Pa LS at 0.25 Pa LS at 0.50 Pa LS at 1.50 Pa C, Control; S, stimulation.
Group F (laminin-coated + microfibers) FC (LS at 0 Pa): control FS at 0.10 Pa FS at 0.25 Pa FS at 0.50 Pa FS at 1.50 Pa
butions of fluid flow, flow velocity, and the magnitude of shear stress on the specimens were calculated and verified. A commercial CFD software, Fluent 6.0 (Fluent, Lebanon, NH, USA), was used for this process. Cell culture with mechanical stimulation The specimens were divided into two groups: only-laminin-coated coverglasses (group L) and laminin-coated coverglasses on which microfibers were deposited (group F). Each group was further divided into five subgroups depending on the magnitude of shear stress applied (Table 1). PC-12 cells were seeded at 2.5× 104 cells/glass on Groups L and F in Petri dishes. The cells were cultured in DMEM-HG containing 10% FBS in an incubator for 2 d. Two days after the seeding, groups L and F were transferred to a flow chamber. Three cover glasses were placed in each flow chamber, and four flow chambers were connected in series. Our preliminary study showed that most cells on the substrates were lost with the application of continuous or intermittent shear stress higher than 1.50 Pa (data are not shown). Then, steady shear stress was applied for 2 h, three times per day for 2 d. To initiate neuronal differentiation while suppressing proliferation, a medium with low-density serum (1% FBS) and nerve growth factor (NGF-7s, 50 ng/ml; Sigma) was added. During the unstressed periods, the medium was circulated at 6.79×10–3 cm/s (shear stress: 2.56 ×10–2 Pa) to refresh the medium and supply sufficient oxygen. Scanning electron microscopy (SEM) of microfiber-based substrates Each sample was fixed in 4% paraformaldehyde for 30 min and washed with phosphate-buffered saline (PBS) several times. Then, the samples were dehydrated in an ethanol/water series (30%, 60%, 70%, 80%, 90%, and 100%) for 1 min each, and air-dried overnight. Each sample was sputter-coated with gold, and their morphology was observed using SEM (JSM-6700F; JEOL, Tokyo) at an acceleration voltage of 5 kV. Double immunostaining To identify differentiated neurons derived from PC-12 cells, cell cultures were double-immunostained with an antibody for the following antigens: NeuN and neuronspecific β-tubulin III. The samples were fixed for 30 min in 4% formaldehyde/PBS. Then, the cells were incubated overnight with a mouse anti-Neuronal nucleus (NeuN) monoclonal antibody (1: 100; Chemicon, Temecula, CA, USA) and a neuronal class III β-tubulin polyclonal antibody (1:2000; Covance, Denver, PA, USA). After the incubation with a primary antibody, the cells were incubated with a secondary antibody, a mixture of mouse anti-goat IgG FITC (1 : 160; Sigma) and Alexa Fluor 594 goat anti-rabbit IgG (1: 300; Molecular Probes, Carlsbad, CA, USA), for 30 min. After mounting them on slides, the cells were examined by fluorescence microscopy using an image analysis system (Axioskop2 Plus; Karl Zeiss, Germany). F-actin staining The samples were rinsed in PBS and fixed in 4% formaldehyde/PBS for 10 min. For F-actin staining, the cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min at 4°C and blocked with 1% bovine serum albumin (BSA)/PBS. Then, the cells were incubated with rhodamine phalloidin (1:100; Molecular Probes) for 20 min at room temperature.
Quantitative analysis of neurite outgrowth and alignment After the neurons were visualized by immunostaining with F-actin, digital images of all the neurons in each group were captured. We assumed that cells having a bipolar or pseudo-unipolar morphology were neurons (14). To determine neurite outgrowth length, a discernible neurite within an image was traced from the soma to the tip of each neurite using the software Image J (ver. 1.33; National Institutes of Health, Bethesda, MD, USA). The average error for the measurement of length was found to be ±2 µm. The numbers of cells used in the measurement of neurite outgrowth were 100–120 in all the groups. The degrees of alignment of PC-12 cells were also analyzed using digital image processing with MATLAB (ver. 6.0; The MathWorks, Natick, MA, USA). The automated image processing method has been used in estimating local directionality and angular deviation (AD) in images of oriented biological tissues and cultured cells (10, 15). In this study, the analysis of each image yielded a distribution of cell orientations, ranging from −90° to 90°, where 0° was defined as the vertical direction. The degree of cell alignment was evaluated by referring to AD, which was calculated using circular statistics, as described by Fisher (16). Then, an accumulator scheme was used to determine the dominant orientation in 40 ×40 pixel sub images. The tested whole image size was in 1280× 1024 pixels and six images were tested in each group for the measurement of alignment. Percentage of cells with neurite outgrowth The cells were photographed using an inverted microscope and examined by counting the cells with neurite outgrowth. When the length of a cell was larger than 1.5-fold the cell body diameter, it was considered as vital cells with neurite outgrowth (17). Six images were captured for each group. The number of cells measured in each image was 80, whereas the number of cells at 1.50 Pa was approximately 20. Then, the percentage of cells with neurite outgrowth was calculated using the following equation. Percentage of cells with neurite outgrowth = number of cells with neurite outgrowth / total cell number Again, the cells regarded as having neurite outgrowth were divided into three subgroups on the basis of neurite outgrowth length. Statistical analysis All the results were evaluated using a one-way analysis of variance (ANOVA), followed by Fisher’s LSD for multiple comparisons. p<0.05 was considered statistically significant. The analysis was conducted using SPSS 11.0 software (SPSS, Chicago, IL, USA).
RESULTS SEM micrographs of microfiber-based substrates revealed that the substrates are composed of fibers with diameters from 3 to 5 µm (Fig. 3A). From the histogram (Fig. 3B), the angular deviation (AD) was calculated as 9.14°. Therefore, the microfibers were expected to serve as a guide for cell outgrowth. We also simulated the internal flow in the chamber using Fluent. The roller pump can adjust flow rate. The range of flow rates applied in this study was 0.52~7.80 ml/min. Figure 4 shows a numerical simulation when the flow rate is 0.85 ml/min. The analytical value and numerical calculation for shear stress was both 0.16 Pa. No vortex formed where shear stress was applied. Therefore, the magnitude of shear stress in each case was confirmed. We confirmed that most PC-12 cells differentiated into neurons by immunocytochemistry using specific neuron cell markers (NeuN and β-Tubulin III) (Fig. 5).
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FIG. 5. Images obtained by double-immunostaining. In both groups, differentiated neurons are identified as those derived from PC-12 cells (×200; bar: 100 µm).
FIG. 3. Microfiber-based substrate: (A) SEM image. (B) Angular distributions of microfibers.
Again, the degree of cellular alignment improved when the shear stress was 0.50 Pa or higher with microfibers (Fig. 7). However, most cells did not align under higher stresses as shown in Fig. 6H and 6J. Regarding to the length of neurite outgrowth, the microfiber-based substrates (group F) were found to be more preferable for neurite outgrowth as expected. This supported the role of microfiber as a guide for neurite outgrowth. Moreover, shear stress also affected the neurite outgrowth. The length of neurite outgrowth was significantly longer in group FS at 0.25 Pa, but decreased at greater shear stresses (Figs. 6 and 8). The percentage of cells with neurite outgrowth changed with the magnitude of shear stress, and there were more neurite outgrowths in group F than in group L (Fig. 9A). Also, the control group (FC), that is, without stimulation, and FS at 0.25 Pa showed higher outgrowth rates than the other groups. However, neurite outgrowths of most cells in FC have outgrowth lengths of less than 80 µm, whereas 50% of those in FS at 0.25 Pa have outgrowth lengths of more than 130 µm (Fig. 9B). DISCUSSION
FIG. 4. Computer simulation for validating uniform shear stress that occur on surface of specimen due to fluid flow. The analytical and numerical calculated shear stresses were both 0.16 Pa.
From the immunostained images, neurite outgrowth was observed and measured by digital image processing. The length of neurite outgrowth in group F was longer than those in group L. Moreover, the neurites in group F were found to be elongated along the direction of the fibers (Fig. 6). In the analyses of cellular orientation or alignment, cells were more highly aligned on fibrous substrates than those without microfibers, that is, the only-laminin-coated glasses.
In this study, we investigated the effects of shear stress and micropatterned fiber substrate on neurite outgrowth and growth guidance. To enhance neurite outgrowth and cell adhesion, we used laminin as baseline coating and added NGF in both groups. Laminin is one of the ECM components that promotes neurite outgrowth in vitro and is present in the areas of axonal branches in vivo (4, 12). NGF is a typical agent that induces the differentiation of PC-12 cells (17, 18). Several investigators have used micropatterned fiber substrates for neurite outgrowth. Miller et al. (19) reported that a micropatterned biodegradable polymer induces the growth orientation of Schwann cells and found that groove width and spacing are important factors in promoting Schwann cell alignment. Similarly, we found that the neurite outgrowth and alignment of PC-12 cells are markedly improved on microfiber-based substrates than on laminin-only-coated glasses. Although there was no significant enhancement of
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FIG. 7. Angular deviation of neurites on two types of substrate along with various shear stresses (six images for each case). In the analyses of cellular orientation, the cells were more highly aligned on the fibrous substrates (groups F) than on the laminin-coated glasses (groups L). Significant differences were observed between groups F and L at each shear stress. In group F, a statistical difference was observed under a shear stress of 0.50 Pa or higher (p<0.05).
FIG. 8. Average outgrowth length of neurites on different substrates along with various shear stresses (n= 100–120, approximately). Significant differences were observed between groups F and L at each shear stress. The average neurite outgrowth length at 0.25 Pa was significantly higher than any other samples in group F (p<0.05).
FIG. 6. Changes in cell morphology depending on magnitude of shear stress and surface morphologies at 4 d after seeding (×100; bar: 100 µm). The neurites in group F were longer than those in group L in terms of the neurite outgrowth length of PC-12 cells. Most cells were detached when the shear stress was higher than 0.50 Pa.
cell alignment on the microfiber-based substrates at two days, the PC-12 cells differentiated into spindle-shaped cells and were highly oriented along the microfibers at 4 d after seeding (Fig. 6). Therefore, the microfiber-based substrate provided a more conducive environment for cell alignment and neurite outgrowth. Mechanical stress is an important component of the host
environment and affects cellular signal transduction and behavior in a various cells. In particular, fluid-induced shear stress was reported to improve cell alignments in various cells (10, 13). This suggests that fluid-induced shear stress, which orients cells via the reorganization of the cytoskeleton, might be applicable for guiding neurite outgrowth. Moreover, the structures of the cell soma and neurites of neurons are associated with the cytoskeleton, which senses mechanical stimuli and generates cellular responses (20). In addition, it has been reported that cell proliferation, migration, and differentiation are controlled via numerous unknown signal pathways connected to the cytoskeleton (6, 20). Therefore, we also adopted fluid-induced shear stress as a stimulus for orienting neurons. When a continuous shear stress was applied for 48 h, most of the cells were detached from the substrates regardless of the magnitude of stress even under 0.50 Pa. Also, the adverse effects of continuous stimuli on cells were reported in previous studies (21, 22). Consequently, we applied a steady shear stress for 2 h, three times a day, for 2 d. Regarding to the alignment of the cell, higher shear
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FIG. 9. Percentage of cells with neurite outgrowth on different substrates and under different shear stresses. (A) Percentage of cells considered to have neural outgrowth within a group. Significant differences were observed between groups F and L at each shear stress. Under the shear stresses of 0.00, 0.25, and 0.50 in group F, the percentage of cells with neurite outgrowth was statistically significant (p<0.05). (B) Distribution of percentage of cell outgrowth based on outgrowth length. Most cell outgrowths in the control group (FC) have outgrowth lengths of less than 80 µm, whereas 50% of those in FS at 0.25 Pa have outgrowth lengths of more than 130 µm (n= 100).
stresses (0.50 Pa or higher) are preferable because smaller angular deviations are induced (Fig. 7). However, many cells detached from substrates at these higher shear stresses as shown in Fig. 6. Moreover, the percentage of cells with outgrowths decreased at higher shear stresses whereas the control group, that is, without stimulation, (FC) showed a higher percentage of cells with under neurite outgrowths (Fig. 9A). Figure 9A is presented on the basis of the assumption that the cells were considered viable and have neurite outgrowths when the length of a cell is larger than 1.5-fold the cell body diameter (17). At this point, we need to further analyze the distribution of the percentage of cells with neurite outgrowths along with the outgrowth length. Figure 9B shows that most cells with neurite outgrowth in control group (FC) have neurite outgrowth length of less than 80 µm, whereas 50% of those in FS at 0.25 Pa have neurite outgrowth length of more than 130 µm, as mentioned before. Figure 8 show that a shear stress of 0.25 Pa with microfibers promotes neurite outgrowth the most. Therefore, the condition of 0.25 Pa shear stress with microfibers is most promising for inducing neurite outgrowth and alignment under the conditions adopted in this study. In this study, the surface pattern of the substrate and the magnitude of shear stress were found to be very important and effective in promoting neurite outgrowth and the align-
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ment of neurons. In particular, there exists a certain magnitude of shear stress that provides the best biomechanical environment with microfibers, that is, 0.25 Pa. This can be explained by cytoskeleton reorganization via the guidance effect of fluid-induced shear stress as described previously. From these results, we conclude that microfiber-based substrates combined with the appropriate fluid-induced shear stress is effective in the functional recovery of nerves, because our model resulted in improved neurite outgrowth and highly controlled alignments, possibly to the desired targets. For the evaluation of the functional recovery of nerves, further research or experimental validation should be performed in animals or clinical studies. However, this study has several limitations: (i) Two-dimensional study of both culture and the alignment of microfibers were performed. (ii) The cells used were PC-12, not primary cultured cells. For three-dimensional structure of a scaffold composed of microfibers, various weaving techniques that are of the conduit type should be studied along with the uniform seeding of cells. In conclusion, the methodologies adopted in this study for providing adjust optimal magnitude of shear stress along with the induction of proper cellular structural morphology proposed a potential for the functional recovery of neurons. Further studies are necessary the limitations of this study mentioned before. ACKNOWLEDGMENTS This work was funded by the “Nano Machining Equipment Development Using Ion Beam” of the Next Generation Technology Development Project of the Ministry of Commerce, Industry and Energy.
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© 2006, The Society for Biotechnology, Japan
Effects of Mechanical Stimuli and Microfiber-Based Substrate on Neurite Outgrowth and Guidance In Ae Kim,1 Su A Park,1 Young Jick Kim,2 Su-Hyang Kim,1 Ho Joon Shin,1 Yong Jae Lee,1 Sung Goo Kang,3 and Jung-Woog Shin1* Department of Biomedical Engineering, Inje University, 607 Eubang-Dong, Gimhae, Gyongnam, Republic of Korea,1 Department of Dental Laboratory Science, College of Health Sciences, Catholic University of Pusan, 9 Bugok 3-dong, Busan, Republic of Korea,2 and School of Biotechnology and Biomedical Science, Inje University, 607 Eubang-Dong, Gimhae, Gyongnam, Republic of Korea3 Received 1 September 2005/Accepted 29 October 2005
We introduced mechanical stimuli and micropatterned substrate with microfibers to investigate their effects on neurite outgrowth along with nerve growth factor in vitro. Two types of surface morphology were used: a surface that was coated by laminin alone and a surface where in microfibers was added on the laminin surface. PC-12 cells were seeded on both surface types and cultured for 2 d. The magnitudes of shear stresses ranged from 0.10 to 1.50 Pa. Two days after stimulation by shear stress, neurite outgrowth and its direction were measured by F-actin staining and digital image processing. When a shear stress of 0.50 Pa was applied, neurons were most highly aligned with microfibers. The average length of neurite outgrowth with microfibers was largest at a shear stress of 0.25 Pa. The results suggest that micropatterned fibers and fluid-induced shear stress are promising for stimulating neurite outgrowth in a desired direction. [Key words: neurite outgrowth length, microfiber-based substrate, PC-12 cells, fluid flow shear stress, electrospinning, nerve regeneration]
tor. When the voltage reaches a critical value, the charge overcomes the surface tension of the deformed drop of a suspended polymer solution formed on the tip of a syringe, and a jet is produced (7, 8). This is a straightforward, costeffective means of producing ultrafine fibers with diameters ranging from microns to a few nanometers. This method requires a small amount of polymer, such as poly(D,L-lactideco-glycolic acid) (PLGA), poly(glycolic acid) (PGA), silk, fibrinogen, collagen, or polymer composites (5, 8). In addition, the electrospun fibrous structure improves cell adhesion, proliferation, and ECM production (6). Another important factor on nerve regeneration is the mechanical environment. The mechanical environment plays a central role in the physiology of various tissues. Shear stress affects both mechanoreception, such as ion channels and integrins/focal adhesions, and response, such as intracellular calcium and nitric oxide production, and cytoskeletal remodeling (9). In particular, fluid-induced shear stress also induces the cellular orientation of osteoblasts, fibroblasts, and endothelial cells. Such stimulation also affects cellular migration and matrix outgrowth (10). Lee et al. (10) observed the effects of shear stress on cell orientation in canine arterial smooth muscle cells (SMCs) and quantified the time and magnitude dependences of shear stress alignment, and found that the alignment of SMCs induced at a high shear stress for longer periods. Klein-Nulend et al. (11) reported that a pulsatile fluid shear stress stimulates osteoblasts and osteocytes differently; when osteoblasts are subjected to a pulsatile fluid flow for 24 h, the cells become
Axons can regenerate spontaneously over relatively short distances (~5 mm), but not over large gaps in the adult peripheral nervous system (PNS). Although regeneration occurs after injury, it does not always result in functional recovery, because regenerating axons are usually directed toward inappropriate targets (1). Therefore, many studies have shown the effects of extracellular matrixes, microgrooved surfaces, new biologically compatible scaffolds, collagen, and fibronectin coating on the growth direction of axons (2–5). In many cell types including neurons, cell survival and growth are influenced by a cell extracellular matrix (ECM) signal. Therefore, various scaffolds based on ECM components, such as laminin or 3D-collagen gels, have been studied (3). However, the directing effects of these structures are very limited. One method that has been attempted is the micropatterning of substrate surfaces. Most micropatterned substrates have been fabricated in the form of microgrooves using ion beams, lasers, or microstamps on which laminin are adsorbed. In addition, it has been reported that neurite guidance and outgrowth are affected by the shapes, dimensions, and raw materials of micropatterned substrates (2–5). Recently, electrospinning has been utilized to produce patterned structures (6). Electrospinning is a fiber manufacturing method that utilizes a high electrical voltage. A high electric field is generated between a polymer fluid contained in a syringe with a capillary tip and a metallic collec* Corresponding author. e-mail: [email protected] phone: +82-55-320-3317 fax: +82-55-327-3292 120
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longer and prostaglandin E2 (PGE2) production increases. However, the effects of shear stress on nerve cells have been rarely reported. Recently, the effects of shear stress on Schwann cells in neurite outgrowth have been investigated. Chafik et al. (12) reported that shear stress is an important component of the natural environment for axon regeneration and that shear stress enhances cellular adhesion, proliferation, and alignment of Schwann cells. In this study, we fabricated microfibers (~3–5 µm in diameter) and deposited them on a substrate parallel to the direction of shear stress. The specific aim of using electrospun micron-sized fibers is to designate a guide for the neurite outgrowth under mechanical stimulation, that is, shear stress compared with other substrates without fibers. The fibers were made smaller than 5 µm considering the cell size and the diameter of neurite. In addition, the small diameter (<5 µm) of the fiber was expected not to disturb the shear stress distribution considering the height of the flow chamber as shown later (Fig. 2). On the basis of the rationale described above, we investigated the effects of shear stress on the neurite outgrowth of PC-12 cells along with changes in the surface morphology of the substrate.
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FIG. 1. Schematic diagram of fluid flow system. The flow system consisted of four consecutive chambers, two reservoirs, and a roller pump.
MATERIALS AND METHODS Preparation of cells and substrate Neurons are more fastidious in their choice of substrate than most other cells. Therefore, a laminin coating was applied for the adhesion of PC-12 cells. Coverglasses (24×36 mm) were coated with a 0.1% laminin solution (0.2 µg/cm2; Sigma, St. Louis, MO, USA) overnight in a petridish, and then rinsed twice with Hank’s Balanced Salt Solution (HBSS). Microfiber-patterned substrates were fabricated by electrospinning. The bulk copolymer of 50 :50 poly(D,L-lactide-co-glycolic acid) (PLGA; Alkermes, Cincinati, OH, USA) was dissolved in a solvent at 40°C to obtain a 50 wt% solution. The solvent consisted of a 1: 1 mixture of N,N-dimethylformamide (DMF; Junsei Chemical, Tokyo) and tetrahydrofuran (THF; Junsei Chemical). For electrospinning, the polymer solution was placed in a 5-ml plastic syringe fitted to an 18-gauge needle. To deposit the aligned microfibers on the laminin surface, a laminin-coated coverglass was glued to a rotating collecting target. To spray the solution, 10 kV was applied between the needle and the collecting target using a high-voltage power supply. The needle was 6.5 cm from the rotating target. The collecting target had a surface velocity of 4.2 m/s. A step motor was used for accurate speed control and the control software used was Turbo C (ver. 3.0; Boland International, Cupertino, CA, USA). Rat pheochromocytoma cells (PC-12) were obtained from the Korean Cell Line Bank (KCLB, accession no. 21721). The cells were cultured in Dulbecco’s modified Eagle medium high-glucose (DMEM-HG), containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT USA), 100 U/ml penicillin (Sigma, St. Louis, MO, USA), and 100 mg/ml streptomycin (Sigma) in an incubator at 37°C with 5% CO2. Design of fluid flow system Figure 1 shows the fluid flow system used for applying shear stress. There are four chambers connected in series, two reservoirs, and a roller pump (Masterflex; Cole-Parmer Instrument Company, Vernon Hills, IL, USA) in the system. One of the reservoirs was open to the atmosphere and the culture medium in the flow circuit was equilibrated with 95% air and 5% CO2 (13). The flow chambers included a flow section 1
FIG. 2. Flow chamber used for applying shear stress: (A) General picture of chamber with cover plate. (B) Schematics of chamber. The flow chamber included a flow section 1 mm in height and 30 mm in width.
mm in height (h) and 30 mm in width (b) (Fig. 2). The pump was controlled using LabVIEW (ver. 6.1i; National Instruments, Austin, TX, USA) using a personal computer. Under the assumption of a Newtonian fluid and fully developed laminar flow, the shear stress applied to the cells was calculated using τ = 6µQ/h2b where µ is dynamic viscosity, Q is flow rate, h is height and b is width. For the calculation, 9.6 ×10–4 Pa⋅s was used as the viscosity (µ) of the culture medium (13). Using computational fluid dynamics (CFD) analysis, we simulated the flow pattern in the fluid chamber. A two-dimensional finite element model was constructed. Using this model, the distri-
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TABLE 1. Classification of experimental groups according to pattern and shear stress magnitude Group L (only laminin-coated) LC (LS at 0 Pa): control LS at 0.10 Pa LS at 0.25 Pa LS at 0.50 Pa LS at 1.50 Pa C, Control; S, stimulation.
Group F (laminin-coated + microfibers) FC (LS at 0 Pa): control FS at 0.10 Pa FS at 0.25 Pa FS at 0.50 Pa FS at 1.50 Pa
butions of fluid flow, flow velocity, and the magnitude of shear stress on the specimens were calculated and verified. A commercial CFD software, Fluent 6.0 (Fluent, Lebanon, NH, USA), was used for this process. Cell culture with mechanical stimulation The specimens were divided into two groups: only-laminin-coated coverglasses (group L) and laminin-coated coverglasses on which microfibers were deposited (group F). Each group was further divided into five subgroups depending on the magnitude of shear stress applied (Table 1). PC-12 cells were seeded at 2.5× 104 cells/glass on Groups L and F in Petri dishes. The cells were cultured in DMEM-HG containing 10% FBS in an incubator for 2 d. Two days after the seeding, groups L and F were transferred to a flow chamber. Three cover glasses were placed in each flow chamber, and four flow chambers were connected in series. Our preliminary study showed that most cells on the substrates were lost with the application of continuous or intermittent shear stress higher than 1.50 Pa (data are not shown). Then, steady shear stress was applied for 2 h, three times per day for 2 d. To initiate neuronal differentiation while suppressing proliferation, a medium with low-density serum (1% FBS) and nerve growth factor (NGF-7s, 50 ng/ml; Sigma) was added. During the unstressed periods, the medium was circulated at 6.79×10–3 cm/s (shear stress: 2.56 ×10–2 Pa) to refresh the medium and supply sufficient oxygen. Scanning electron microscopy (SEM) of microfiber-based substrates Each sample was fixed in 4% paraformaldehyde for 30 min and washed with phosphate-buffered saline (PBS) several times. Then, the samples were dehydrated in an ethanol/water series (30%, 60%, 70%, 80%, 90%, and 100%) for 1 min each, and air-dried overnight. Each sample was sputter-coated with gold, and their morphology was observed using SEM (JSM-6700F; JEOL, Tokyo) at an acceleration voltage of 5 kV. Double immunostaining To identify differentiated neurons derived from PC-12 cells, cell cultures were double-immunostained with an antibody for the following antigens: NeuN and neuronspecific β-tubulin III. The samples were fixed for 30 min in 4% formaldehyde/PBS. Then, the cells were incubated overnight with a mouse anti-Neuronal nucleus (NeuN) monoclonal antibody (1: 100; Chemicon, Temecula, CA, USA) and a neuronal class III β-tubulin polyclonal antibody (1:2000; Covance, Denver, PA, USA). After the incubation with a primary antibody, the cells were incubated with a secondary antibody, a mixture of mouse anti-goat IgG FITC (1 : 160; Sigma) and Alexa Fluor 594 goat anti-rabbit IgG (1: 300; Molecular Probes, Carlsbad, CA, USA), for 30 min. After mounting them on slides, the cells were examined by fluorescence microscopy using an image analysis system (Axioskop2 Plus; Karl Zeiss, Germany). F-actin staining The samples were rinsed in PBS and fixed in 4% formaldehyde/PBS for 10 min. For F-actin staining, the cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min at 4°C and blocked with 1% bovine serum albumin (BSA)/PBS. Then, the cells were incubated with rhodamine phalloidin (1:100; Molecular Probes) for 20 min at room temperature.
Quantitative analysis of neurite outgrowth and alignment After the neurons were visualized by immunostaining with F-actin, digital images of all the neurons in each group were captured. We assumed that cells having a bipolar or pseudo-unipolar morphology were neurons (14). To determine neurite outgrowth length, a discernible neurite within an image was traced from the soma to the tip of each neurite using the software Image J (ver. 1.33; National Institutes of Health, Bethesda, MD, USA). The average error for the measurement of length was found to be ±2 µm. The numbers of cells used in the measurement of neurite outgrowth were 100–120 in all the groups. The degrees of alignment of PC-12 cells were also analyzed using digital image processing with MATLAB (ver. 6.0; The MathWorks, Natick, MA, USA). The automated image processing method has been used in estimating local directionality and angular deviation (AD) in images of oriented biological tissues and cultured cells (10, 15). In this study, the analysis of each image yielded a distribution of cell orientations, ranging from −90° to 90°, where 0° was defined as the vertical direction. The degree of cell alignment was evaluated by referring to AD, which was calculated using circular statistics, as described by Fisher (16). Then, an accumulator scheme was used to determine the dominant orientation in 40 ×40 pixel sub images. The tested whole image size was in 1280× 1024 pixels and six images were tested in each group for the measurement of alignment. Percentage of cells with neurite outgrowth The cells were photographed using an inverted microscope and examined by counting the cells with neurite outgrowth. When the length of a cell was larger than 1.5-fold the cell body diameter, it was considered as vital cells with neurite outgrowth (17). Six images were captured for each group. The number of cells measured in each image was 80, whereas the number of cells at 1.50 Pa was approximately 20. Then, the percentage of cells with neurite outgrowth was calculated using the following equation. Percentage of cells with neurite outgrowth = number of cells with neurite outgrowth / total cell number Again, the cells regarded as having neurite outgrowth were divided into three subgroups on the basis of neurite outgrowth length. Statistical analysis All the results were evaluated using a one-way analysis of variance (ANOVA), followed by Fisher’s LSD for multiple comparisons. p<0.05 was considered statistically significant. The analysis was conducted using SPSS 11.0 software (SPSS, Chicago, IL, USA).
RESULTS SEM micrographs of microfiber-based substrates revealed that the substrates are composed of fibers with diameters from 3 to 5 µm (Fig. 3A). From the histogram (Fig. 3B), the angular deviation (AD) was calculated as 9.14°. Therefore, the microfibers were expected to serve as a guide for cell outgrowth. We also simulated the internal flow in the chamber using Fluent. The roller pump can adjust flow rate. The range of flow rates applied in this study was 0.52~7.80 ml/min. Figure 4 shows a numerical simulation when the flow rate is 0.85 ml/min. The analytical value and numerical calculation for shear stress was both 0.16 Pa. No vortex formed where shear stress was applied. Therefore, the magnitude of shear stress in each case was confirmed. We confirmed that most PC-12 cells differentiated into neurons by immunocytochemistry using specific neuron cell markers (NeuN and β-Tubulin III) (Fig. 5).
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FIG. 5. Images obtained by double-immunostaining. In both groups, differentiated neurons are identified as those derived from PC-12 cells (×200; bar: 100 µm).
FIG. 3. Microfiber-based substrate: (A) SEM image. (B) Angular distributions of microfibers.
Again, the degree of cellular alignment improved when the shear stress was 0.50 Pa or higher with microfibers (Fig. 7). However, most cells did not align under higher stresses as shown in Fig. 6H and 6J. Regarding to the length of neurite outgrowth, the microfiber-based substrates (group F) were found to be more preferable for neurite outgrowth as expected. This supported the role of microfiber as a guide for neurite outgrowth. Moreover, shear stress also affected the neurite outgrowth. The length of neurite outgrowth was significantly longer in group FS at 0.25 Pa, but decreased at greater shear stresses (Figs. 6 and 8). The percentage of cells with neurite outgrowth changed with the magnitude of shear stress, and there were more neurite outgrowths in group F than in group L (Fig. 9A). Also, the control group (FC), that is, without stimulation, and FS at 0.25 Pa showed higher outgrowth rates than the other groups. However, neurite outgrowths of most cells in FC have outgrowth lengths of less than 80 µm, whereas 50% of those in FS at 0.25 Pa have outgrowth lengths of more than 130 µm (Fig. 9B). DISCUSSION
FIG. 4. Computer simulation for validating uniform shear stress that occur on surface of specimen due to fluid flow. The analytical and numerical calculated shear stresses were both 0.16 Pa.
From the immunostained images, neurite outgrowth was observed and measured by digital image processing. The length of neurite outgrowth in group F was longer than those in group L. Moreover, the neurites in group F were found to be elongated along the direction of the fibers (Fig. 6). In the analyses of cellular orientation or alignment, cells were more highly aligned on fibrous substrates than those without microfibers, that is, the only-laminin-coated glasses.
In this study, we investigated the effects of shear stress and micropatterned fiber substrate on neurite outgrowth and growth guidance. To enhance neurite outgrowth and cell adhesion, we used laminin as baseline coating and added NGF in both groups. Laminin is one of the ECM components that promotes neurite outgrowth in vitro and is present in the areas of axonal branches in vivo (4, 12). NGF is a typical agent that induces the differentiation of PC-12 cells (17, 18). Several investigators have used micropatterned fiber substrates for neurite outgrowth. Miller et al. (19) reported that a micropatterned biodegradable polymer induces the growth orientation of Schwann cells and found that groove width and spacing are important factors in promoting Schwann cell alignment. Similarly, we found that the neurite outgrowth and alignment of PC-12 cells are markedly improved on microfiber-based substrates than on laminin-only-coated glasses. Although there was no significant enhancement of
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FIG. 7. Angular deviation of neurites on two types of substrate along with various shear stresses (six images for each case). In the analyses of cellular orientation, the cells were more highly aligned on the fibrous substrates (groups F) than on the laminin-coated glasses (groups L). Significant differences were observed between groups F and L at each shear stress. In group F, a statistical difference was observed under a shear stress of 0.50 Pa or higher (p<0.05).
FIG. 8. Average outgrowth length of neurites on different substrates along with various shear stresses (n= 100–120, approximately). Significant differences were observed between groups F and L at each shear stress. The average neurite outgrowth length at 0.25 Pa was significantly higher than any other samples in group F (p<0.05).
FIG. 6. Changes in cell morphology depending on magnitude of shear stress and surface morphologies at 4 d after seeding (×100; bar: 100 µm). The neurites in group F were longer than those in group L in terms of the neurite outgrowth length of PC-12 cells. Most cells were detached when the shear stress was higher than 0.50 Pa.
cell alignment on the microfiber-based substrates at two days, the PC-12 cells differentiated into spindle-shaped cells and were highly oriented along the microfibers at 4 d after seeding (Fig. 6). Therefore, the microfiber-based substrate provided a more conducive environment for cell alignment and neurite outgrowth. Mechanical stress is an important component of the host
environment and affects cellular signal transduction and behavior in a various cells. In particular, fluid-induced shear stress was reported to improve cell alignments in various cells (10, 13). This suggests that fluid-induced shear stress, which orients cells via the reorganization of the cytoskeleton, might be applicable for guiding neurite outgrowth. Moreover, the structures of the cell soma and neurites of neurons are associated with the cytoskeleton, which senses mechanical stimuli and generates cellular responses (20). In addition, it has been reported that cell proliferation, migration, and differentiation are controlled via numerous unknown signal pathways connected to the cytoskeleton (6, 20). Therefore, we also adopted fluid-induced shear stress as a stimulus for orienting neurons. When a continuous shear stress was applied for 48 h, most of the cells were detached from the substrates regardless of the magnitude of stress even under 0.50 Pa. Also, the adverse effects of continuous stimuli on cells were reported in previous studies (21, 22). Consequently, we applied a steady shear stress for 2 h, three times a day, for 2 d. Regarding to the alignment of the cell, higher shear
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FIG. 9. Percentage of cells with neurite outgrowth on different substrates and under different shear stresses. (A) Percentage of cells considered to have neural outgrowth within a group. Significant differences were observed between groups F and L at each shear stress. Under the shear stresses of 0.00, 0.25, and 0.50 in group F, the percentage of cells with neurite outgrowth was statistically significant (p<0.05). (B) Distribution of percentage of cell outgrowth based on outgrowth length. Most cell outgrowths in the control group (FC) have outgrowth lengths of less than 80 µm, whereas 50% of those in FS at 0.25 Pa have outgrowth lengths of more than 130 µm (n= 100).
stresses (0.50 Pa or higher) are preferable because smaller angular deviations are induced (Fig. 7). However, many cells detached from substrates at these higher shear stresses as shown in Fig. 6. Moreover, the percentage of cells with outgrowths decreased at higher shear stresses whereas the control group, that is, without stimulation, (FC) showed a higher percentage of cells with under neurite outgrowths (Fig. 9A). Figure 9A is presented on the basis of the assumption that the cells were considered viable and have neurite outgrowths when the length of a cell is larger than 1.5-fold the cell body diameter (17). At this point, we need to further analyze the distribution of the percentage of cells with neurite outgrowths along with the outgrowth length. Figure 9B shows that most cells with neurite outgrowth in control group (FC) have neurite outgrowth length of less than 80 µm, whereas 50% of those in FS at 0.25 Pa have neurite outgrowth length of more than 130 µm, as mentioned before. Figure 8 show that a shear stress of 0.25 Pa with microfibers promotes neurite outgrowth the most. Therefore, the condition of 0.25 Pa shear stress with microfibers is most promising for inducing neurite outgrowth and alignment under the conditions adopted in this study. In this study, the surface pattern of the substrate and the magnitude of shear stress were found to be very important and effective in promoting neurite outgrowth and the align-
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ment of neurons. In particular, there exists a certain magnitude of shear stress that provides the best biomechanical environment with microfibers, that is, 0.25 Pa. This can be explained by cytoskeleton reorganization via the guidance effect of fluid-induced shear stress as described previously. From these results, we conclude that microfiber-based substrates combined with the appropriate fluid-induced shear stress is effective in the functional recovery of nerves, because our model resulted in improved neurite outgrowth and highly controlled alignments, possibly to the desired targets. For the evaluation of the functional recovery of nerves, further research or experimental validation should be performed in animals or clinical studies. However, this study has several limitations: (i) Two-dimensional study of both culture and the alignment of microfibers were performed. (ii) The cells used were PC-12, not primary cultured cells. For three-dimensional structure of a scaffold composed of microfibers, various weaving techniques that are of the conduit type should be studied along with the uniform seeding of cells. In conclusion, the methodologies adopted in this study for providing adjust optimal magnitude of shear stress along with the induction of proper cellular structural morphology proposed a potential for the functional recovery of neurons. Further studies are necessary the limitations of this study mentioned before. ACKNOWLEDGMENTS This work was funded by the “Nano Machining Equipment Development Using Ion Beam” of the Next Generation Technology Development Project of the Ministry of Commerce, Industry and Energy.
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