Nerve tissue model on a micropatterned surface: Axon guidance and neural regeneration

Abstract

This study focuses on the design, production and testing of a micropatterned PDMS surface, featuring micropillars and microchannels to study the regeneration of individual axons of PC12 nerve cells after injury. Micropillar organization on the surface was designed to restrict the PC12 cell bodies while axons were guided into microchannels, allowing observation of individual axons. Surfaces were coated with poly(L-lysine) to improve cell attachment and proliferation. Netrin-1, a chemoattractant molecule and axonal elongation enhancer, was introduced in a gelatin methacrylate (GelMA) hydrogel carrier at the opposite end of the channels. Schwann cells (SC) were co-cultured with PC12 cells to enhance axon extension. MTT and Live-Dead assays showed 90% viability of the PC12 and Schwann cells on surfaces. The average PC12 axon length in the channels was 51 ± 19 μm; which increased to 75 ± 16 μm and 177 ± 31 μm upon co-culture with Schwann cells and Netrin-1 incorporation along with co-culturing, respectively, showing their synergistic effect on axon elongation. To study axon damage and regeneration processes, PC12 axons extended into the microchannels were cut using a microtome blade. An increase in the expression of injury markers ATF3, GFAP and S100β was observed after the injury with confocal microscopy, and their decrease from days 14 to 21 indicated the initiation of axon regeneration. The platform consisting of patterned PDMS surface, Schwann cells and Netrin-1 holds potential as a valuable tool for nerve damage and repair studies, and for in vitro testing of novel nerve tissue engineering strategies.

Highlights

• A novel micropatterned platform was designed for neural axon guidance and regeneration studies.

• Axons of PC12 neural cells were directed into microchannels.

• Presence of Schwann cells and chemoattractant Netrin-1 enhanced axon extension.

• Regeneration of severed axons were detected by the expression intensity of the injury markers ATF3, GFAP and S100β.

Introduction

Nerve damage resulting from accidents or injuries present a significant clinical challenge. Peripheral nerve injuries are observed in approximately 2.8% of trauma patients. As described by Seddon and Sunderland, advanced stages of nerve injury can lead to severe consequences [1, 2]. Repair of damaged nerves in affected individuals not only enhances their quality of life but also mitigates potential economic losses due to workforce depletion. Consequently, researchers in nerve tissue engineering and regenerative medicine fields have been intensively investigating novel strategies to address nerve damage, particularly over the past two decades [3].

In the treatment of nerve injuries, the initial intervention, depending on the severity of the damage, is the end-to-end suturing technique [4]. However, for a successful application of this method, the length of the injury must be less than 1 cm. In cases of more extensive injuries, alternative treatment methods are required. One established treatment approach for such injuries, which is considered the gold standard, involves the use of autografts derived from the patient’s own nerve tissue [5]. Nonetheless, the limited regeneration potential and sensory loss associated with harvesting tissue from the donor site, have prompted research into alternative treatments [6, 7].

Alternatives to autografts are allografts and xenografts (tissues sourced from other humans and animals, respectively), but they present disadvantages, including risks of immune reaction, infection, tissue rejection, and limited availability of suitable donor tissues [8]. Such limitations with the traditional treatment methods, and the necessity to repair or replace damaged tissues and restore their function, led to the emergence of tissue engineering and regenerative medicine fields [9, 10]. Tissue engineering aims to create living, three-dimensional tissues and organs using specific combinations of materials, bioactive agents, cells, and signaling molecules, bringing together fundamental scientific knowledge and engineering approaches [11]. Artificial nerve grafts, called nerve guidance conduits, are tissue engineering products that aim to overcome the shortcomings of traditional treatments for nerve damage, in order to improve the outcome of regenerative processes after nerve injury [3].

Research on nerve conduits is primarily conducted using two-dimensional (2D) in vitro cell cultures, followed by in vivo testing in animal models. However, in vitro studies do not fully replicate the physiological conditions of the human body. In in vivo applications, sensory loss may occur in surviving animals, or the subjects may require euthanasia which is not acceptable from the ethical point of view. Literature indicates that although in vivo animal models better represent human neurological functions and healing processes than in vitro studies, they still have limitations [12]. One significant limitation is the species difference; animals may respond to treatments or diseases in ways that are not entirely applicable to humans. Additionally, the controlled environment of laboratory settings can fail to replicate the complex interactions found in human physiology, which can lead to discrepancies in results. The variability in genetic backgrounds, age, and environmental factors among animal subjects can introduce inconsistencies that may not accurately reflect human conditions. There are also ethical concerns, such as the use of animals in research raises questions about welfare and the moral implications of subjecting living beings to experimental procedures [13].

Numerous variables must be considered during the planning phase for utilizing an artificial nerve conduit system in in vivo experiments [14]. These include the type (neurapraxia, axonotmesis, and neurotmesis) and extent of the nerve injury to be induced, the size of the animal subjects which definitely affect the applicability of the conduit, and the differences in the healing processes between the animal models and humans. For example, the “rat sciatic model” is frequently employed for testing nerve conduits due to its cost effectiveness, resistance of rats to surgical site infections and capacity for conducting electrophysiological studies [15–17]. However, these models are limited to short nerve injuries and making physiological comparisons of damage and healing processes to those in humans is not possible. For instance, the duration required for complete recovery of injured nerves in rats is shorter (around 4 weeks) than that in humans (up to 2 years), complicating the interpretation of experimental outcomes [18]. Given the physical dissimilarities of experimental animals to humans and ethical considerations, the use of tissue engineering products and tissue models is favored in preliminary testing phases [19–22].

Various microfluidic systems and 3D tissue models have been developed to investigate nerve injury treatments for tests conducted under in vitro and in vivo conditions [23, 24]. In a microfluidic primary culture model designed by Southam et al. collagen surfaces consisting of two compartments linked with channels were used. With this model, axons of nerve cells seeded in one compartment were able to extend into the second compartment and branch out. “Campenot Chamber,” was another system employed in a number of studies, particularly in investigating the effects of viruses on axon elongation [25–29].

Despite their availability, such systems generally lack the ability to apply controlled injury or fail to offer sufficient resolution to track axon regeneration at the single cell level. Moreover, neuron-glia interactions in regionally restricted environments remain poorly understood due to the lack of glial cell incorporation with good spatial control with respect to the axons. These gaps emphasize the need for simplified yet functional in vitro systems that allow precise axon patterning, localized injury, and real time monitoring of regeneration. In this study, this challenge is addressed by developing a micropatterned platform that combines unidirectional axon guidance with spatially confined Schwann cell interaction and direct injury to axons. Guided by the accumulated expertise of our group regarding cell behavior on micropatterned surfaces, we designed a novel platform consisting of a surface with different patterned regions, aiming to sort and restrict cell bodies in one place while letting axons extend anisotropically. On this platform it was possible to introduce different agents that promote axon elongation such as Netrin-1 into separate compartments of the model to act specifically on the PC12 cell bodies or the axons, perform injury to the axons extended, study markers of axonal damage, and observe the subsequent regeneration of the axons.

The surface is decorated with two distinct patterns, micropillars and microchannels, with the pillar configuration varying in different regions (Fig. 1). After cell seeding, cell bodies were confined within the micropillar region, the organization of which directed the axons towards microchannels and lead them to extend within the channels. In order to enhance guidance and extension of axons into the channels, a chemotropic agent, Netrin-1, loaded in a GelMA hydrogel carrier was positioned at the region opposite to the cell bodies. In order to mimic the composition of natural nerve tissue, Schwann cells (SC) were seeded atop the microchannels, while the axons were extending underneath within the channels. After achieving sufficient extension of axons through the channels, a cut was made with extreme care, using microtome blades. The damage and healing process was assessed with confocal microscopy, through the expression of selected injury marker molecules ATF3, GFAP, and S100Β. A key distinction of our study from existing literature is the open design, with pillars and channels to sort and isolate cell bodies and axons, which enabled us to study a single axon before and after the damage during the regeneration process.

Fig. 1.

Design and production of the PDMS surface. a 3D model of the micropatterned surface, b Negative photolithography wafer mold, c Micropatterned PDMS surfaces prepared using the mold

Materials and methods

Fabrication of the silicon wafer and micropatterned PDMS surfaces

The three dimensional micropatterned surface was designed using AutoCAD 2019 (Autodesk Inc., USA) (Fig. 1a). The design consists of three distinct regions on a PDMS surface.

Region A serves as the cell seeding site, region B1 and B2 contain two sets of micropillar patterns, and region C consists of microchannels (Fig. 1). Region B, positioned after the cell seeding chamber, has pillars strategically arranged in two sections with different spacings between them, to sort cells and separate the cell bodies. These pillars also guide the axons toward the microchannels. The design of the channels in region C aims to ensure that the axons of the cells from the preceding compartment extend in a unidirectional manner. This configuration was designed to facilitate microscopic observation of the repair processes of individual axons following damage.

The micropatterned surface was fabricated first by creating a negative mold on a photoresist coated silicon wafer, through traditional photolithography. A silicon wafer (diameter 10 cm) was spin coated with SPR-220-3 positive photoresist at 1000 rpm. The photoresist coated silicon wafers were then aligned with a photomask of the micropattern design and subjected to UV exposure (365 nm). The patterns formed on the photoresist surface were developed through immersion in a developer bath and wafer was then hard baked, yielding the photoresist negative molds. A silanization process was then conducted using trichloro(1,1,2,2-perfluorooctyl)silane. Finally, a mixture of PDMS prepolymer and crosslinker (10:1 ratio, Sylgard 184 Silicone Elastomer Kit, Dow Chemical Company, USA) was poured onto the photoresist mold, maintained under vacuum at 60 °C for 3 h, resulting in the formation of surfaces covered with micropatterns, 4.8 μm in height (Fig. 1b, c). The surfaces were UV sterilized and coated with poly(L-lysine) (1 mg/mL in syringe filtered sodium borate buffer, 1 mM, pH 8.5) under sterile conditions, to increase cell attachment to the PDMS substrate. Coated surfaces were kept sterile until use.

Characterization of micropatterned PDMS surfaces

Scanning electron microscopy (SEM)

Micropatterned PDMS surfaces were coated with gold under vacuum using a sputter coater and examined with a scanning electron microscope (SEM, Quanta 400, FEI, USA).

Profilometry

Micropatterned surfaces were analyzed using a profilometer (S Neox, Sensofar, Spain). Topographical images obtained were used to assess the homogeneity of the poly(L-lysine) coating and changes in surface dimensions.

Fourier Transform Infrared Spectroscopy (FTIR)

In order to confirm the presence of the poly(L-lysine) coating, micropatterned surfaces were analyzed with FTIR spectroscopy (Spectrum 100, Perkin-Elmer, USA), and the resulting spectrum was compared with that of poly(L-lysine) reported in literature.

Sterilization of the micropatterned platforms

After the production of PDMS micropatterned surfaces, both sides of the surfaces were exposed to UV for 15 min in a laminar flow cabinet for sterilization prior to cell culture studies. Additionally, the antibacterial properties of sodium borate buffer used in the poly(L-lysine) coating contributed to the sterility of the surfaces.

In vitro studies

PC12 and Schwann cell (SC, S42) culture and seeding on micropatterned surfaces

Sterile, poly(L-lysine) coated, micropatterned surfaces were used in in vitro testing. All experiments and analyses were conducted with at least three biological replicates. The PC12 cell line was cultured in T75 flasks in Ham’s F12 medium supplemented with 15% DHS, 10% FBS, 1% penicillin-streptomycin, and 50 ng/mL NGF, at 37 °C in a 5% CO₂ incubator. Cells were harvested via trypsinization and collected by centrifugation at 3000 rpm for 5 min. The resulting cell pellet was resuspended in fresh medium and seeded onto the cell seeding region ‘A’ of the micropatterned surfaces (Fig. 1a), at a density of 3×10³ cells/surface. After attachment, cells were incubated in medium containing 90% reduced serum (1% FBS in culture medium). Schwann cells were maintained in DMEM high medium containing 5% FBS and 1% penicillin/streptomycin. Cells were detached from flasks by trypsinization, centrifuged at 3000 rpm for 5 min, and the resulting cell pellet was resuspended in the medium. 3×10³ cells were seeded in region C of the surfaces (Fig. 1a). For the co-culture of PC12 and Schwann cells, DMEM-F12 medium containing 1.5% DHS, 1% FBS, 1% penicillin-streptomycin, and NGF (50 ng/mL) was used.

MTT and Live/Dead cell viability assay

The Live/Dead cell viability assay was performed according to the manufacturer’s protocol (ThermoFisher Scientific). Briefly, cells cultured on micropatterned surfaces were stained at different time points (days 1, 7, and 14) with calcein AM (2 µM) and ethidium homodimer-1 (4 µM) dyes for 20 min at room temperature. The surfaces were then washed with PBS and studied with a confocal laser scanning microscope (CLSM) (Zeiss LSM 800, Germany). The MTT cell viability assay was performed on days 1, 3, and 7 of cell culture. Cells in TCPS wells served as positive controls, while cell-free surfaces were used as negative controls. At each time point, the medium was removed, MTT solution was added, and incubated for 3 h at 37 °C in a humidified 5% CO₂ incubator. Then, 4% HCl in isopropanol was added to dissolve the formazan crystals formed. After 1 h of incubation at room temperature with gentle shaking, the absorbance was measured at 550 nm using a UV-Vis spectrophotometer (Thermo Scientific, Multiscan Spectrum with Cuvette, USA). Cell numbers were determined using a calibration curve previously constructed (n = 3).

DAPI/Phalloidin staining

Cells cultured on the micropatterned surfaces were fixed with 4% (v/v) paraformaldehyde for 15 min at room temperature followed by washing with PBS. The fixed cells were permeabilized with 0.1% Triton X-100 (in PBS) for 5 min and blocked with 1% BSA (in PBS, w/v) for 30 min at 37 °C. Cells were then stained with FITC-conjugated Phalloidin probe (for 1 h at 37 °C) and DAPI (for 20 min at room temperature), washing with PBS in between dyes. Dyed samples were studied with a CLSM (Leica DM2500, Germany), and the micrographs were analyzed using NIH ImageJ software.

Addition of the chemoattractant Netrin-1

In order to optimize the Netrin-1 release from GelMA, a model molecule, BSA, which has a similar molecular weight (Netrin-1 80 kDa, BSA 70 kDa) and a comparable 3D structure, was used as a model molecule in release studies. FITC-labeled BSA (200 ng) was added to a 20% GelMA solution (150 μL). Photoinitiator (Irgacure 2959, 1% (w/v)) was employed to crosslink the GelMA solution by UV exposure (365 nm, 5 s) in a 96 well plate. To obtain the release profile of BSA-FITC (Netrin-1 model), the samples were placed in 5 mL of PBS in a shaking incubator at 37 °C. The amount released into the buffer was determined for up to 48 h using fluorescence spectroscopy (Molecular Devices, SpectraMaxM2). After optimizations with BSA, Netrin-1 (200 ng/sample) was loaded into the GelMA hydrogels, which were then placed at the end of region C of the micropatterned surface.

Schwann Cell co-culture and Netrin-1 treatment

Effect of Netrin-1 and Schwann Cell (SC) co-culture on axonal lengths of PC12 cells were examined under CSLM. Three different experimental groups were prepared; Group 1: PC12 only; Group 2: PC12 co-cultured with SC; Group 3: PC12 co-cultured with SC in the presence of Netrin-1. For each experimental group, 3 biological replicates were prepared, and at least 10 distinct axonal protrusions were measured from each replicate from different regions of interest, with a total of 35 measurements per experimental group. Outliers were assessed and excluded from statistical analysis using the Robust Regression and Outlier Removal (ROUT) method (Q = 1%) in GraphPad Prism (v. 8.0.2) software. After determination and exclusion of outliers, descriptive statistics were obtained using the same software. Statistical comparisons between experimental groups were performed using one-way ANOVA with multiple comparisons. Differences were considered statistically significant for a p-value equal to or less than 0.05 (p ≤ 0.05).

Microscopical examination of axonal damage

Performing axonal damage

Axonal damage was applied manually using a cryomicrotome blade on day 7 of cell culture in a laminar flow cabinet under stereomicroscope. In order to ensure reproducibility of the injury across samples, specific visual reference points on the micropatterned surfaces were used to align the blade and guide the location and orientation of the cut. The blade was applied perpendicularly across the channel entrance, targeting the midpoint of axonal extensions. The procedure was performed by two trained researchers and in order to minimize variability between experimental sets and operators. Each operator practiced extensively on separate test surfaces before carrying out the cuts with extreme care. Moreover, each experimental set was assigned to a single operator. Post injury axonal behavior was consistently observed using live cell imaging and CLSM, confirming the reproducibility of injury patterns across replicates. The growth of the axons in the medium containing Netrin-1 was monitored with the CLSM Live Imaging System for 24 h.

Immunofluorescence assay

PC12 and Schwann cells were cultured under optimized medium conditions and in the presence of Netrin-1 (Ham’s F12 medium containing 200 ng/mL NGF and 90% reduced serum) for 7 days. Following axonal damage, at several time points (Days 1, 7, 14, 21) the cells cultured on micropatterned surfaces were fixed and stained for neuronal damage markers. Briefly, the cells were fixed with 4% (v/v) paraformaldehyde for 15 min at room temperature followed by washing with PBS. Fixed cells were permeabilized using Triton X-100 (0.1% v/v in PBS, 10 mM, pH 7.4) and kept in a BSA solution (2% w/v, in PBS, 10 mM, pH 7.4) for 30 min at 37 °C for blocking non-specific binding. The cells were then incubated in primary antibody solutions (rabbit anti-ATF3 antibody, diluted 1:200 in PBS; goat anti-GFAP antibody, diluted 1:200 in PBS; mouse anti-S100-B antibody, diluted 1:200 in PBS; mouse/rabbit anti-βIII Tubulin antibody, diluted 1:200 in PBS) overnight at 4 °C, secondary antibody solutions (goat anti-rabbit antibody, Alexa Fluor 594; rabbit anti-goat antibody, Alexa Fluor 488; donkey anti-mouse antibody, Alexa Fluor 647, all diluted 1:100 in PBS) for 2 h at room temperature, and DAPI (1:1000 in PBS, 10 mM, pH 7.4) for 20 min at room temperature, respectively, washing with PBS in between steps. Stained samples were examined with CLSM, and semi-quantitative imaging analysis was performed on micrographs using NIH ImageJ software. Marker expression was compared between time points using one-way ANOVA with Bonferroni’s post-hoc testing, and differences were considered statistically significant for p ≤ 0.05.

Statistical analysis

All characterization and in vitro studies were performed in triplicates unless otherwise stated. After calculating arithmetic means and standard deviations, significant differences between mean values in control and test groups were determined using a one-tailed Student’s t-test or one-way ANOVA with multiple comparisons, utilizing standard software in Microsoft Excel or the GraphPad Prism (v. 8.0.2) software. Differences were considered statistically significant for p-values equal to or less than 0.05.

Results and discussion

Characterization of micropatterned surfaces

Micropatterned PDMS surfaces were coated with poly(L-lysine) in order to enhance cell attachment and examined before and after coating process by SEM and profilometer as well as FTIR spectroscopy.

1. SEM and Profilometer Analyses

   SEM images of the micropatterned surfaces demonstrated that the pillars and channels were homogeneously organized in accordance with the intended configuration. Poly(L-lysine) coating was also found to be uniform across surfaces (Fig. 2). Surfaces were analyzed with a profilometer to verify that the pattern dimensions were produced as designed (Fig. 2b). Micropillar dimensions and the spacing between them in regions B1 and B2, as well as the channel dimensions in region C, were produced according to the design specifications. After poly(L-lysine) treatment, the coating thickness on the surfaces was measured at three different regions, and the average coating thickness was found as 0.135 ± 0.045 µm. This indicated that the micropatterned surfaces were coated with poly(L-lysine) without significantly altering the dimensions or spacings of the micropatterns.

2. Fourier Transform Infrared (FTIR) Analyses

Fig. 2.

Characterization of the micropatterned PDMS surfaces with 4.8 µm tall pillars. a SEM micrograph of PDMS surface, b Profilometry of PDMS surface, c TIR spectra of PDMS surfaces with and without poly(L-lysine) coating

The characteristic infrared (IR) peaks of PDMS were studied on both poly(L-lysine) coated and uncoated surfaces. Peaks observed were: 789–796 cm⁻¹ (-CH₃ rocking and Si-C stretching in Si-CH₃), 1020–1074 cm⁻¹ (Si-O-Si stretching), 1260–1259 cm⁻¹ (-CH₃ deformation in Si-CH₃), and 2950–2960 cm⁻¹ (asymmetric CH₃ stretching in Si-CH₃) (Fig. 2c) [30]. Additionally, on the poly(L-lysine) coated surface, characteristic amide I and III peaks, indicative of proteins, proved the presence of poly(L-lysine) coating [31].

Viability of PC12 and Schwann cells

The viability of cells cultured on poly(L-lysine) coated PDMS surfaces were determined with Live-Dead cell viability and cytotoxicity testing. Images obtained with CLSM from three different regions were processed with NIH Image J, and PC12 cell viability was found as 95.02 ± 0.78% on day 1, 96.46 ± 0.01% on day 3, and 96.29 ± 0.01% on day 7 (Fig. 3a), indicating no cytotoxic effect from the substrate, as expected. There was a noticeable increase in the number of protrusions extended from the cells on Day 7 (Fig. 3a), suggesting that the poly(L-lysine) coating improved cell affinity to the PDMS surfaces.

Fig. 3.

Live-Dead analysis on poly(L-lysine) coated PDMS surfaces on Day 7. a PC12 cells, b Schwann cells. Live: Calcein-AM, green. Dead: Ethidium Homodimer-1, red/yellow. Scale bar: 50 μm

MTT assay results demonstrated that the cells cultured on the surfaces for 1, 3, and 7 days were able to attach and proliferate. The absorbance values obtained were converted into viable cell counts using a calibration curve established with PC12 cells of the same passage number (Fig. 4). Cell numbers were observed to have increased 6-fold within 7 days of culture.

Fig. 4.

MTT cell proliferation assay for PC12 and Schwann cells on poly(L-lysine) coated PDMS surfaces. a PC12 cells, b Schwann cells. Statistical differences (* p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Schwann cell (SC) viability on poly(L-lysine) coated PDMS surfaces was also investigated. Attachment and proliferation of SC were monitored through confocal microscopy and MTT assay on days 1, 3, and 7 (Fig. 3b and Fig. 4). On day 1, microscopy images showed that the cells adhered to the surface and displayed healthy morphology. By day 3, the number of SC had increased approximately 2-fold, indicating significant proliferation, which was supported by MTT assay results, showing an increase in metabolic activity (Fig. 4b). By day 7, the cell population had grown nearly 5-fold compared to day 1, with cells covering a large portion of the PDMS surface. The behavior of SC observed on poly(L-lysine) coated PDMS surfaces is consistent with the general understanding of cell-material interactions, where surface properties such as roughness and patterning can significantly influence cell growth and differentiation [32]. These results are in line with previous studies that demonstrate the cell adhesion and proliferation on poly(L-lysine) modified biomaterial surfaces, such as PDMS, which is known for its biocompatibility and flexibility, making it suitable for neural tissue engineering [33].

Preliminary investigations on the interaction of PC12 cells with micropatterned surfaces showed that a rapid increase in cell population adversely affected our analyses of axonal damage and healing processes. On the other hand, we observed that lowering the cell density (by seeding less cells) prevented robust cell-cell interactions, and as a result, cell viability. Therefore, a number of experiments were performed to optimize the number of cells seeded onto the surfaces as well as the composition culture media. The serum used in culture media serves as a crucial nutrient source as it is rich in growth factors, components affecting proliferation and adhesion, as well as hormones, lipids, and minerals. Serum starvation is a method widely used in molecular biology for various purposes, including synchronization of the cell cycle, inhibition of cell proliferation, induction of autophagy, acceleration of metabolic processes, and promotion of differentiation [17, 34–38]. We, therefore, studied the effect of different serum concentrations (full serum, 50% reduced, 90% reduced, and serum-free) on PC12 cell proliferation and axonal extension. Cell culture experiments conducted in TCPS multi-well plates showed that cells in serum-free medium could not maintain their viability (data not shown). This loss of viability was attributed to the serum-free medium’s lack of nutrition required for metabolic functions [39]. Conversely, PC12 cells cultured in media with reduced serum content were able to adhere to the surfaces, and the decrease in proliferation rate was proportional to the reduction in serum concentration, while the axon lengths increased (data not shown). This increase in axon lengths was consistent with the literature, which suggests that cells experiencing serum starvation may be directed to differentiation rather than proliferation, which supported by the presence of NGF, resulted in longer axons [34].

We determined that the optimal serum concentration for suppressing cell proliferation while maximizing axon length was 90% less than that of the original composition. Changes in NGF concentration (25 or 50 ng/mL) on the other hand, did not result in a significant difference in axon lengths of cells cultured in media containing 90% reduced serum. Based on these results, PC12 cells were cultured in 90% reduced serum media supplemented with 50 ng/mL NGF for subsequent in vitro studies. Throughout the culture period, monitoring the initial cell number on the micropatterned surfaces led us to the optimal cell seeding density of 3000 cells per surface. Under these conditions, PC12 cells cultured on micropatterned surfaces were fixed on days 7 and 14, and then followed by staining of their cytoskeletons and nuclei, with subsequent examination using CLSM. It was noted that in one case a cell seeded in region ‘A’ traversed region ‘B1’, successfully reached region ‘B2’, and extended towards the channels in region ‘C’ as intended (Fig. 5a–c). Under optimal culture conditions, PC12 cells were observed to extend their axonal projections into the microchannel region C, achieving axon lengths of up to 88 µm by day 14.

Fig. 5.

Cell-surface interactions of the PC12 and SC. a Schematic representation of PC12 cells on the surfaces between the pillars and their axons extended in the channels, b PC12 cells on Day 7, c PC12 cells on Day 14, d Schematic representation of SC on the surfaces along with PC axons extended in the channels, e Interaction between PC12 cells and SC on Day14. PC12 cells or their axons are in the channels and the SC over the channel walls. Culture medium with NGF: 50 ng/mL, Serum: 90% reduced. Cell cytoskeleton (AlexaFluor-488 Phalloidin, green), nucleus (DAPI, blue). Scale bar: 20 µm

In order to study the behavior of SC on micropatterned surfaces, these cells were cultured in the same medium composition, and their morphologies were examined with CSLM. SC were seeded in region C, which is the microchannel patterned part of the surface (Fig. 5d). It was expected that SC would wrap the extended axons and form myelin coats around them. SC showed a tendency to grow and form aggregates while remaining on top of the channels and the neurite extensions (Fig. 5e). The SC formed a network on the micropatterned surface, adhering the surface and proliferating without entering the channels, thus achieving the desired surface interaction in the study. When SC on the microchannels were studied with CLSM, it was observed that the optimized medium composition supported spreading of SC (Fig. 5e).

Effect of Netrin-1 on axon extension

Netrin-1 was incorporated into GelMA hydrogel in order to achieve a slow release, to guide neurite outgrowth and support nerve regeneration. Before conducting experiments with Netrin-1, fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) was used as a model protein to optimize the release conditions for efficient delivery by fine-tuning hydrogel properties. BSA was chosen due to its similar molecular weight and three-dimensional structure to Netrin-1, making it a suitable substitute [40, 41].

To evaluate protein loading and release, BSA-FITC was added to a 5% (w/v) GelMA solution at concentrations of 50, 100, and 200 ng/mL. The mixture was then crosslinked under UV light to form hydrogels, which were subsequently immersed in isotonic phosphate buffered saline (PBS) solution to assess protein release. GelMA hydrogels without BSA served as controls. Due to the high equilibrium water content of the 5% GelMA hydrogel ( ≥ 90%), nearly all the encapsulated BSA was released within the first 10 min, indicating rapid release (data not shown).

To achieve a more sustained release profile, the crosslinking density of the hydrogel was increased by modifying three key parameters: (1) the GelMA concentration was raised from 5 to 20% (w/v), (2) the crosslinking agent (Irgacure) concentration was increased from 0.5 to 1% (w/v), and (3) the UV crosslinking time was extended from 10 to 20 s. Under these conditions, BSA release from the hydrogel was significantly prolonged, extending beyond 48 h (Fig. 6a).

Fig. 6.

Effect of Netrin-1 release on axon lengths in the absence and presence of Schwann cells. a Release of BSA-FITC (model molecule used to represent Netrin-1 in release studies) from GelMA hydrogel, b effect of SC and Netrin-1 on axon length. Statistical analysis of axon length measurements under different conditions. Statistical differences (* p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

The effect of SC co-culture and Netrin-1 release from GelMA hydrogel on PC12 axon length was investigated for 7 days. PC12 axon lengths were measured for each experimental group (PC12, PC12 + SC, PC12 + SC+Netrin-1) using CLSM. Distinct axonal protrusions of at least 10 different cells from 3 different regions of interest were measured, with a total of 35 measurements for each experimental group. The data obtained was put through outlier analysis in GraphPad Prism (version 8.0.2) software using the Robust Regression and Outlier Removal (ROUT) method (Q = 1%). After elimination of outliers, the mean axon length values were found as 51.43 ± 18.57, 74.86 ± 15.56, and 177.00 ± 31.24 for PC12, PC12 + SC, and PC12 + SC+Netrin-1 groups, respectively (Table 1). While a statistically significant positive effect on axon extension was observed for PC12 cells when co-cultured with Schwann cells, the synergistic effect of Netrin-1 and SC presence was found to be more pronounced, compared to SC co-culture alone (Fig. 6b). This finding is consistent with the existing literature, where the role of Schwann cells in promoting axonal growth and the enhancement provided by Netrin-1 both as a guidance cue and a growth promoter in neural development is well documented [42]. These outcomes suggest our platform is suitable for further studies investigating the synergistic effects of Schwann cells and Netrin-1 in axonal regeneration and guidance, while providing a rational basis for hypothesizing that co-culture conditions and chemotactic agents can be strategically used to improve axonal growth after injury [43, 44].

Table 1.

Descriptive statistics of axon length measurements of the experimental groups on Day 14

Group	PC12	PC12 + SC	PC12 + SC+Netrin-1
Mean Axon Length (µm)	51 ± 19	75 ± 16	177 ± 31
Min. Axon Length (µm)	30	57	141
Max. Axon Length (µm)	88	115	256

Study of axon damage and regeneration

After incubation of PC12 cells on micropatterned surfaces for 7 days, the cells were subjected to damage by making a vertical cut using a microtome blade. After damaging, cut cells were fixed and stained for the cytoskeleton and nuclei (Fig. 7). The damaged cells (shown with red arrows in Fig. 7a) continued to live and formed growth cones at the cut site, indicating it is possible to monitor the damage site on the platform to track the progress of regenerating axons, as designed. This was further confirmed by live imaging of PC12 cells for 24 h after performing axonal damage (Fig. 7b). CLSM images obtained every 15 min were combined in a video that shows the re-extension of a cut axon (Supplementary Data). The PC12 cell damaged (Fig. 7b, indicated by a red circle) is seen to be extending its axon beyond the cut site at the end of 24 h. This was interpreted as an indication of regeneration of the damaged PC12 cell, in the presence of Netrin-1 and Schwann cells.

Fig. 7.

CLSM images of PC12 cells after injury on surfaces. a 5 days post-damage. b PC12 cell images taken every 15 min for 24 h after damage (All the images are also presented as a supplement video). Red arrows indicate growth cones in a and the cut sites in b. Red circles indicate the growing axon after damage. Cytoskeleton (Alexa Fluor-488 Phalloidin, green), nucleus (DAPI, blue)

Immunofluorescence assay

Seven days after co-culture of PC12 and SC, the effect of the damage created in the axonal extensions of the PC12 cells were studied through examination of the expression of injury markers S100β, ATF3 and GFAP for 21 days (Fig. 8). The expression levels of ATF3 and S100β proteins on days 1 and 7 after damage were higher than that observed on days 14 and 21, and also that of the control group (Fig. 8a, b). It was reported in the literature that ATF3 protein is induced in peripheral neurons in response to injury, supports regeneration, and is therefore considered a marker of nerve damage [45]. Similarly, it was demonstrated that expression of S100β protein significantly increased after a nerve injury, promoting the formation of neuronal extensions and stabilization of axonal structures, thereby supporting nerve repair [46]. Therefore, the increases observed in ATF3 and S100β protein expressions on days 1 and 7 were outcomes expected of a nerve damage. These results also show that it was possible to apply and detect nerve damage using our platform. On days 14 and 21, the expression levels of these proteins showed a significant decrease. These findings suggest that ATF3 and S100β proteins play a significant role in the early response to nerve damage, and their expression decreases over time in parallel with the start of regeneration, as reported in the literature [47]. The observed temporal pattern of ATF3 and S100β protein expression highlights their critical involvement in the early response, during the acute phase of nerve injury. Their significant increase on days 1 and 7 is in accordance with their roles in neuronal regeneration and axonal stabilization, as supported by previous studies [48, 49]. This pattern highlights the dynamic nature of the nerve injury response, where initial repair mechanisms shift to processes focused on long term stabilization and functional recovery [47].

Fig. 8.

Effect of damage of the axons of PC12 cells on the expression levels of injury protein markers, S100β (purple), ATF3 (green), GFAP (green) calculated by NIH Image J. The graphs of fluorescence/cell vs. time and CSLM images of protein markers a S100β protein, b ATF3 protein and c GFAP and β Tubulin III (red). Results are shown as means ± SD, comparisons tests were performed by one-way ANOVA with Bonferroni’s post-hoc. *p < 0.05, **p < 0.01 and ***p < 0.001

Staining of another injury marker, glial fibrillary acidic protein (GFAP), was applied together with that of β-III-tubulin to study morphological changes and the healing process of the neural cells (Fig. 8c). The co-cultured PC12 and SC cells were shown to align initially on the surface along the microchannels, influenced by the surface topography, then spread as their number increased. PC12 cell numbers were seen to be significantly affected by the damage induced by the microtome blade. On day 1 post-damage, the cell density was markedly reduced compared to the undamaged control group. Cells increase their number on the surface over the 21 days following the injury. On day 7, a slight increase in cell density and organization was observed. Microtubules, as dynamic components of the cytoskeleton, are critical for maintaining cell shape and structural integrity. The increase in β-III-tubulin signal (red) on day 7 indicates that the microtubule structures of the cells were getting reorganized and that the cellular skeleton was beginning the healing process. It was observed that the cells were aligned along the channels on day 7, with this orientation becoming further prominent by day 14. Expression levels of GFAP per cell did not present a statistically significant change for these time points (Fig. 8c). On day 21, significant increases in cell density and organization were demonstrated, with no notable increase in GFAP expressions compared to days 7 and 14. GFAP is an important protein found in glial cells that supports cellular structure, enhancing cell resilience and stability [50]. It is a protein upregulated in many types of Schwann cells upon dedifferentiation from the myelinating phenotype, in response to nerve injury [51]. The steady levels of expression of GFAP in SC cultured on our platform could be explained with the fact that these cells did not assume a myelinating phenotype prior to the damage performed on axons. As a contributor to cytoskeleton assembly and maintenance, a steady expression of GFAP per SC in our culture was maintained as cells continued to proliferate, while no change in phenotype occurred during the damage and healing process that required a change in its expression profile. These results suggest that ATF3 and S100ß are more suitable markers in the monitoring of axonal damage and regeneration processes on our platform.

The use of PC12 cells in this study, while methodologically advantageous, poses a clear limitation in terms of physiological and translational relevance. PC12 cells do not form fully mature axons, lack synaptic structures, and originate from a tumor lineage, which can introduce genetic and functional differences compared to primary neurons [52]. As such, they cannot fully mimic the in vivo neuronal response to injury or regeneration. Nonetheless, PC12 cells remain a widely accepted first line model for axon guidance and regeneration studies due to their robust differentiation in response to NGF and consistent behavior on engineered surfaces [53, 54]. These properties make them particularly suitable for early phase testing of novel micro engineered systems. However, in order to make a reliable assessment of the translational potential of the platform, it is crucial to use primary neuronal cultures (dorsal root ganglion or cortical neurons) or neural progenitor cells, which mimic the human nervous system more closely. These cell types allow in vitro evaluations of axonal behavior, glial interactions, and injury response upon damage that more accurately reflect those in vivo.

Compared to existing platforms, the system presented offers several unique advantages. Microfluidic systems, while highly precise in gradient generation and compartmentalization [55, 56], are often limited by complex multilayer fabrication, susceptibility to leakage, and challenges in applying localized mechanical injury. Additionally, their closed architecture can restrict access for direct intervention to cells or impose difficulties in high resolution imaging. On the other hand, 3D nerve conduits and hydrogels excel in mimicking extracellular matrix environments and are well suited for in vivo implantation [57], but do not provide the spatial control or optical transparency required for real time, single axon level analysis. The approach in this study presents an advantage by enabling unidirectional axon guidance, controlled axotomy, and quantitative regenerative tracking in a system that is optically accessible, easy to fabricate and handle, and open to modification. It is particularly suitable for early phase mechanistic studies of regeneration and neuron-glia interactions. One key feature that distinguishes our platform from many existing axonal guidance models is the spatial separation between neuronal extensions and Schwann cells. In our design, axons are physically guided to grow within microchannels, while Schwann cells remain on the surface of the device, without settling into the channels. This creates a controlled interface where neuron-glia interactions can be studied at defined contact points rather than across diffuse and overlapping regions. Generally, such spatial resolution is not attained in similar microfluidic systems or 3D constructs. This arrangement also allows for a more precise analysis of how localized glial presence influences axonal regeneration.

Conclusion

The aim of this study was to design a novel chip with micropatterned surface to study the performance of nerve cells, their orientation, neurite extension, and healing behavior after giving a damage to their axons. As part of the conducted studies, micropatterned surfaces containing different regions with micropillars and microchannels were produced. The physical and chemical properties of these surfaces were examined before and after poly(L-lysine) coating. Their interactions with nerve cells and Schwann cells were comprehensively investigated. SEM and profilometer analyses of the surfaces demonstrated that the micropatterns could be produced homogeneously in the designed dimensions. After the poly(L-lysine) coating, the micropatterned structure of the surfaces was preserved, with an average coating thickness of 0.135 ± 0.045 µm. It was shown that PC12 and SC had over 90% viability on the poly(L-lysine) coated surfaces. One of the main objectives of the study was, to guide and direct the axonal extensions of the PC12 cell along the microchannels, while the main cell bodies are trapped at a certain region of the designed model. Schwann cells cultured on the surfaces did not settle into the channels but proliferated while adhering to the surface. Moreover, Schwann cells clustered on the nerve cells oriented along the microchannels, to enhance regeneration, as expected and given in literature [26, 33, 58].

At a certain point of culturing, axonal extensions of PC12 cells were damaged using a microtome blade by extreme care. Following the application of cuts to the axons, an increase in the expression of axonal injury markers, ATF3 and S100Β proteins, was observed. This increase confirmed that the axon damage was induced. On day 14 and 21, the expression of ATF3 and S100 proteins decreased, indicating that the cells entered the regeneration phase. During the recovery process, live cell imaging studies demonstrated that the axons could extend and maintain their orientation along the microchannels. However, only a portion of the PC12 cell bodies could be retained by the micropillars; contrary to the goal of directing only the axonal extensions into the channels. Some cells exceeded the micropillar region and transitioned into the microchannel area, suggesting design optimization regarding pattern height, and also the neural cell model utilized in the study. PC12 cells used in this study are immortalized cells isolated from adrenal gland tumors (pheochromocytoma). In previous studies conducted by our group using various micropatterned surfaces, it was observed that cancer cells with metastatic capacity could take advantage of the flexibility of their nuclei to settle in the gaps between microchannels and micropillars while healthy cells could not [59–61]. Therefore, it is hypothesized that PC12 cells may possess similar nuclear plasticity, leading to less than ideal cell body restriction in the micropillar region. It is also anticipated that this goal could be achieved when using healthy neural progenitor cells, neural stem cells, or primary neural cell cultures instead of tumor-derived cell lines.

Based on the findings of this study, the platform we developed shows significant potential as a tool for studies involving axon damage and regeneration. Future studies utilizing healthy neural progenitor cells, neural stem cells, or primary neural cultures could further enhance the applicability of this system for neural repair and regeneration research.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1007/s10856-025-06959-3.