Guiding cellular behavior with micro/nanostructured cellulose acetate substrates fabricated using femtosecond laser lithography

Abstract

The lack of efficient, reliable, and sustainable substrates for directed cell growth and tissue engineering remains a critical challenge in modern biomedical applications. Conventional cell culture platforms, such as flat and featureless surfaces, fail to replicate the complex microenvironments necessary for guiding cellular behavior, including contact guidance. This limitation is particularly significant in applications such as skeletal muscle tissue engineering, where the alignment and morphology of cells play a critical role in functionality. Additionally, the growing demand for eco-friendly materials necessitates the development of renewable and biocompatible alternatives to synthetic polymers. To address these challenges, we have fabricated micro- and nanopatterned substrates on cellulose acetate, a sustainable and biodegradable biopolymer, using a femtosecond laser. Ultrafast laser processing enables precise patterning of cellulose surfaces to achieve isotropic and anisotropic features of physiological relevance. Substrates with anisotropic grooves were evaluated for their ability to support and guide the alignment of mouse myoblast cells (C2C12), with the results demonstrating the potential of these engineered patterned substrates in supporting cell attachment, proliferation, and alignment. This work not only shows the potential of using cellulose acetate as an advanced cell culture platform, but also highlights femtosecond laser processing as a versatile tool for creating substrates that mimic natural extracellular environments. By addressing the key limitations of current cell culture methods, this study advances the development of sustainable, functional scaffolds for tissue engineering and regenerative medicine applications.

---

1. Introduction

The development of advanced engineered biomaterials capable of regulating cellular behaviour and guiding cell fate has received significant attention in the recent past.¹ Mimicking the in vivo microenvironment, comprising both biochemical and physical cues, is essential for regulating cellular behaviour, organization, and fate.² Thus, developing sustainable micro- and nanostructured substrates that replicate the physical characteristics of native tissues and extracellular matrix (ECM) is crucial for applications in tissue engineering, micro-implant surgery, and drug delivery.³ However, conventional cell culture platforms, such as flat polystyrene and glass surfaces, lack the necessary topographical cues to direct cellular organization, leading to cellular behavior that differs from the physiological conditions. Addressing these limitations requires biomimetic substrates that better recapitulate the native cellular environment.

Various micro- and nanofabrication techniques such as photolithography, soft lithography, and electrospinning have been widely used to fabricate topographical cues that mimic in vivo like features.⁴ These topographical cues influence cellular behavior such as adhesion, proliferation, migration and differentiation.⁵ Different micro/nanopatterned substrates that promote contact guidance – a phenomenon where cells orient or migrate along topographical features – are well reported in the literature.^6,7 Cellular alignment is one of the important phenomena observed in anisotropic tissues such as muscles, nerves, and tendons. This has been extensively studied in vitro, where structural organization is vital for proper function.⁸ For instance, nano/microgrooves of varying sizes,⁹ nanopillars,¹⁰ and aligned nanofibers¹¹ have been widely used to achieve cellular alignment using the aforementioned techniques. However, these techniques face challenges, including the need for high-cost infrastructure, photomasks, and compatibility with a limited range of photoactive synthetic materials.¹² Additionally, achieving high resolution, reproducibility, and scalability remains a persistent issue, limiting their broader application in the development of biomaterials.¹³

Femtosecond laser (fs)-based fabrication has recently gained attention as an advanced tool for engineering micro- and nanostructures on biomaterial surfaces with unparalleled precision and minimal thermal damage.^14,15 Unlike traditional laser processing techniques, femtosecond lasers operate on ultrafast timescales, enabling localized material modification without affecting the bulk properties of the substrate.¹⁶ Ultrashort pulse laser assisted fabrication of different micro/nanostructures on metals such as stainless steel, titanium, and gold, and polymers such as polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), polyvinyl alcohol (PVA), polystyrene, and polydimethyl siloxane (PDMS), for regulating cellular behavior has been reported in previous studies.¹⁷ For instance, short pulsed F2 laser ablated line and square microstructures were shown to tune the adhesion of mouse fibroblast cells on PTFE.¹⁸ Boker et al. demonstrated the fabrication of periodic nanostructures on stainless steel and titanium using picosecond (ps) laser beam interference ablation, which led to reduced adhesion and osteogenic differentiation in human mesenchymal stem cells (hMSCs), thus inhibiting osseointegration of implants and promoting easy removal of implants after healing.¹⁹ In another study, the wettability of patterned PDMS was controlled by changing the pulse energy of the fs laser during ablation. The patterned structures showed adhesion of C2C12 cells with cellular alignment.²⁰ Although these materials have been widely used for fabricating implants and tissue engineering applications, the corrosion issues of metals, allergies, susceptibility to infections and low biocompatibility are some of their limitations.¹⁹ Micro/nanopatterning of biopolymers enables the creation of intricate surface features that mimic the native ECM, eliminating the above limitations and providing critical guidance cues for cells. This emphasizes the need for biopolymer-based engineered substrates.

Cellulose, a naturally abundant and biodegradable biopolymer, offers a sustainable alternative to synthetic polymers due to its biocompatibility, mechanical strength, and ease of chemical modification.²¹ Among its derivatives, cellulose acetate (CA), obtained by the acetylation of hydroxyl groups, has garnered great attention in various biomedical applications such as drug delivery, wound healing, and tissue engineering.²² Its improved solubility, processability and surface tunability make it an attractive material for the development of advanced cell culture substrates.²³ However, the fabrication of precise and reproducible micro- and nanostructures on cellulose-based materials to create anisotropic surfaces suitable for cellular guidance has been a bottleneck. The ability to fabricate well-defined anisotropic patterns on cellulose derived materials using fs laser presents an opportunity to develop innovative cell culture platforms that can direct cell growth and improve tissue organization.

In this study, we report the fabrication of micro/nanopatterned cellulose acetate substrates using a fs laser. These micro/nano patterned substrates were used for cell culture applications. By culturing muscle cells on these substrates, we evaluated their biocompatibility and analyzed their cell behavior through morphological assessments. These findings demonstrate that the engineered cellulose substrates influence cellular responses, helping in cellular growth and alignment, providing insights into their potential use as advanced cell culture platforms. This work aims to bridge sustainable material engineering with biomedical innovation, paving the way for eco-friendly and high-performance substrates in tissue engineering and other biological applications.

2. Materials and methods

2.1. Materials

Cellulose acetate and dimethyl formamide (DMF, analytical grade) were purchased from Sigma Aldrich. Mouse myoblast cells (C2C12) used in this study were kindly gifted by CCMB, Hyderabad. Growth media, fetal bovine serum (FBS), antibacterial-antimycotic (anti-anti), and trypsin were purchased from HiMedia and Gibco.

2.2. Fabrication of cellulose acetate patterned substrates

Cellulose acetate (CA) solution (10% w/v) was prepared in dimethyl formamide (DMF). The solution was mixed well and sonicated for 1 h until a clear, viscous solution was visible. A thin film was prepared on a glass coverslip using a spin coater (SpinNXG-P1) at 4500 rpm for 1 min and substrates were patterned using a fs laser beam. Briefly, the coverslip with a thin cellulose acetate film was adjusted on the stage of the microscope with an attached CCD camera and exposed to a laser beam (Coherent: Chameleon, Ultra I). Lithography was performed using a fs laser beam, operating at 800 nm wavelength, 140 fs pulse duration. The beam was tightly focused onto the polymer film using a 100× objective (0.9 NA). The fabrication parameters were optimized by varying the laser power and the writing speed. Anisotropic grooves and isotropic mesh patterns were fabricated and observed under an optical microscope (Olympus Infinity1).

2.3. Characterization

The detailed morphological features of the fabricated patterned substrates were investigated using scanning electron microscopy (SEM, FEI Quanta 200). The substrates were sputter coated with platinum and imaged under a high vacuum at 15 kV. The linewidth of the structures was determined from the SEM micrographs using image analysis software, Fiji (ImageJ). A commercial atomic force microscope (AFM, Asylum/Oxford Instruments, MFP3D Origin), equipped with a standard tip on a cantilever beam, was used to investigate the surface roughness and the topography of the patterned substrate in tapping mode at ambient temperature at a scan rate of 0.30 Hz. Analysis of the depth of the laser exposed region and average roughness was calculated using GWYDDION 2.60. The emission spectrum of the patterned CA substrate was obtained using a confocal laser scanning microscope (LSM 780, Carl Zeiss). A Plan-Apochromat 40×/1.3 NA (oil) objective was used for focusing the laser beams (405 nm, 488 nm and 561 nm) onto the sample and image acquisition was done using the Zen 2012 software. Surface functionalization of the substrate after fs laser exposure was examined using Raman spectroscopy (Witec 300 RAS confocal Raman system) and X-ray photoelectron spectroscopy (XPS, Kratos Analytical, AXIS Supra). To determine the hydrophilicity/hydrophobicity of the patterned substrates, wettability tests were performed using a contact angle setup (Apex). The water contact angle of the fabricated structures with the respective control films was measured using software (ACAM) at a time interval of 1 s (n = 10 images).

2.4. Cell culture

Mouse myoblast cells (C2C12, ATCC-CRL-1772) cultured in high glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 20% (v/v) fetal bovine serum (FBS) (HiMedia, RM9955), 1% glutamax (Gibco, 35050) and 1% anti-anti (HiMedia, A002) were used to check the response on the fabricated micro/nanostructured substrates. The cells were cultured in T25 culture flasks (Tarson) and incubated at 37 °C in a humidified atmosphere with 5% CO₂ (Thermo). The cell doubling time under these conditions was observed to be around 24 hours. The growth medium was changed every 48 h and the cells were harvested/subcultured at 70% confluency using 0.5% trypsin-EDTA (HiMedia – TCL003).

2.5. Cytocompatibility and morphological observations

The cytocompatibility of the fabricated structures was evaluated using C2C12 cells. The structures were UV sterilized for 60 min and washed with sterile Dulbecco's phosphate buffered saline (DPBS). The structures were coated with collagen I (25 μg mL⁻¹) and incubated overnight at 4 °C followed by DPBS washing. The cells were then seeded on the structures at a seeding density of 2000 cells per well and checked for adhesion and growth after 24 h incubation. A glass coverslip was used as a control. The experiment was performed in duplicate. Calcein-AM (Invitrogen) and Hoechst 33342 (Invitrogen, H3570) staining were performed to assess cell viability. Briefly, media from all the wells were pulled down, to which Calcein (1 : 2000) and Hoechst (1 : 5000) were added. 1 mL of media was added to each well and incubated for 15 min in a CO₂ incubator. After incubation, the medium was removed and fresh medium was added. The plate was then observed under a fluorescence microscope (EVOS FL Auto). The morphological features of cells growing on the patterned substrates, such as projection area, circularity, aspect ratio and orientation angle, were analysed using ImageJ (Fiji) software. The morphology of the cells on the patterned substrates was also observed using SEM.

2.6. Analysis of cellular orientation and nuclear morphology

The cellular orientation on the anisotropic patterned substrates was evaluated to study the cellular alignment. Briefly, the angle of the nucleus orientation with respect to the groove direction in patterned CA and with respect to the x-axis in flat substrates was measured using ImageJ and was used to study the cellular orientation. Cells were defined as oriented or aligned along the patterned substrate if the angle was less than 20°. Nuclear features such as area, aspect ratio and circularity were also evaluated to understand the effect of the pattern on nuclear morphology. A flat CA film was used as the control.

2.7. Analysis of actin expression and orientation

Actin staining was carried out to check the expression of actin and orientation of actin stress fibers. Briefly, the cells were cultured on a patterned substrate for 24 h and then fixed with 4% paraformaldehyde in DPBS for 15 min. After fixation, permeabilization was done by adding 0.1% Triton-X 100 (Sigma Aldrich) and incubated for 10 min. After permeabilization, the cells were washed with DPBS and treated with 4% BSA (prepared in DPBS) for 45 min, followed by staining with Actin-Phalloidin Alexa fluor 532 (1 : 500) and Hoechst (1 : 5000). The samples were incubated for 2 h at room temperature, followed by three DPBS washes. The samples were then mounted on a glass slide with mounting media (ProLong, Thermo Fisher) and sealed with Eu kit (Sigma Aldrich). The slides were then observed in 40× (oil immersion) under a confocal microscope to check actin expression and coherency (LSM 780, Carl Zeiss).

2.8. Statistical analysis

For statistical analysis, 50 cells from each sample were used (N = 3) and analysed by Fiji (ImageJ) and a Student's t-test was applied to check statistical significance. Results of p < 0.05 were considered significant (p < 0.05 denoted by *, p < 0.01 denoted by **, p < 0.001 denoted by *** and non-significant denoted by ns).

3. Results and discussion

3.1. Fs laser patterning of cellulose acetate (CA)

A thin film of cellulose acetate (CA) was prepared using the optimized concentration of 10% CA in DMF for fs laser-assisted surface patterning. The use of CA enables thin film formation more effectively due to its superior solubility compared to pristine cellulose. Direct laser writing was performed to create a patterned substrate by focusing the fs laser beam on a spin-coated thin film of CA. Fig. 1A shows a schematic of fs laser patterning on cellulose acetate. Anisotropic grooves and isotropic mesh of varying features were fabricated and observed under an optical microscope (Fig. S1). The use of fs laser lithography provides high resolution, precision, reproducibility, maskless processing and versatility with sustainable materials such as cellulose acetate, unlike conventional techniques that require photoresins, masks and sophisticated infrastructure.²⁴

Fig. 1 (A) Schematic showing fs laser patterning on CA; optimization of the fabrication parameters: (B) SEM micrographs showing change in linewidth with increase in laser power at a constant speed of 120 μm s⁻¹ (scale bar: 5 μm), (C) power vs. linewidth plot, (D) SEM micrographs showing change in linewidth with increase in speed at a constant power of 500 mW (scale bar: 5 μm) and (E) speed vs. linewidth plot.

Systematic studies were carried out to determine the optimum fabrication parameters such as laser power, writing speed, and their influence on the morphological features of the micro/nanostructured patterns. For laser power optimization, patterning was performed at a constant speed of 120 μm s⁻¹ by varying the laser power in the range of 120 mW to 800 mW. With an increase in the laser power, the pattern linewidth increased from 510 nm at 120 mW to 1538 nm at 800 mW as shown in Fig. 1B. Below 120 mW, no patterning was observed on the CA film whereas above 600 mW, no significant change was observed in linewidth (Fig. 1C). At the lowest power of 120 mW, a linewidth of 510 nm was obtained; however, the lines were discontinuous and non-uniform, attributed towards insufficient laser power for uniform patterning. The best resolution of 1000–1100 nm was achieved by varying the power from 300 to 500 mW with a uniform and fine pattern without any defects. For writing speed optimization, at a constant laser power of 500 mW, the scanning speed was varied from 20 to 500 μm s⁻¹. The fabricated micro/nanostructures were continuous and uniform at lower scanning speeds, whereas they were discontinuous and uneven at higher scanning speeds. It was observed that with an increase in speed, the linewidth decreased (Fig. 1D). This can be attributed to the exposure time and different number of laser pulses incident per unit area on the CA film at varying scanning speeds.²⁵ The best linewidth of 1000 nm was obtained at a speed of 120 μm s⁻¹ (Fig. 1E). Based on the optimization results, an average laser power of 500 mW and writing speed of 120 μm s⁻¹ were used for the efficient fabrication of patterned substrates. These optimized parameters are crucial for ensuring the high reproducibility and resolution of the patterned substrates.

3.2. Characterization of the patterned CA substrate

Fig. 2A shows the SEM micrographs of fabricated anisotropic microgrooves (i–ii) and isotropic mesh (iii–iv) patterns in CA film, confirming high-resolution patterning with uniform and fine features. No defects were observed in the films in regions adjacent to the laser-exposed area, and the linewidth in each pattern was found to be around 1000 ± 100 nm without any modification of the polymer solution. This is better as compared to the previously reported resolution of 5 μm²⁶ and 17 μm²⁷ achieved using fs laser patterning of cellulose. It was also observed that the features of the pattern, such as linewidth, depth and roughness, can be controlled by tuning the laser parameters such as power and writing speed. This highlights the advantage of femtosecond (fs) laser-based fabrication, as it is a non-thermal process that reduces material damage. This is because, unlike traditional laser ablation, fs lasers excite surface electrons into a plasma state without causing heat-induced melting or evaporation.^28,29 The real time fabrication of the patterned substrate is shown in Video S1.

Fig. 2 Morphological characterization of the fabricated patterned substrates: (A) SEM micrographs showing anisotropic grooves of (i) 3 μm and (ii) 5 μm ridge size and isotropic mesh of (iii) 3 μm and (iv) 5 μm separation (scale bar: 20 μm) and (B) AFM of patterned substrates showing surface roughness and depth profile (i–iii).

The surface topography of the patterned CA substrates was analyzed using AFM (Fig. 2B(i)). A detailed 3D view of the morphology of the anisotropic groove pattern is shown in Fig. 2B(ii), which indicates the effect of laser exposure near the borders. The non-ablated region retained the morphology of the original CA coating and was not affected by laser exposure. The average roughness of the structures was found to be around 133 nm. From the depth profile graph (Fig. 2B(iii)), it is evident that the groove depth (∼450 nm) was uniform throughout the pattern.

Confocal laser scanning microscopy (CLSM) imaging of patterned CA showed fluorescence after fs laser patterning, indicating the material-laser interactions. The patterned CA exhibited excitation-dependent emission upon exposure to different lasers at 405 nm, 488 nm, and 561 nm (Fig. 3A). No fluorescence was observed in the flat CA film (Fig. S2). This could be due to in situ synthesis of carbon quantum dot-like materials due to ultrafast laser exposure.³⁰ The surface wettability test of the patterned and flat CA confirmed the hydrophilic nature of the surfaces, with water contact angles of 58.3° and 55.6° for flat and patterned substrates, respectively (Fig. 3B(i and ii)). The slight decrease of ∼3° in the water contact angle for the patterned CA can be attributed to the introduction of surface functionalities due to laser exposure (Fig. 3B(iii)).²⁶

Fig. 3 (A) Fluorescence images of the patterned substrates (scale bar: 50 μm): (i) phase and under different excitation wavelengths (ii) 405 nm, (iii) 488 nm, and (iv) 561 nm; (B) surface wettability of (i) flat and (ii) patterned substrates showing hydrophilic nature.

The Raman spectra of the patterned and flat CA film are shown in Fig. 4A. Patterned CA showed characteristic D and G bands at 1363 cm⁻¹ and 1590 cm⁻¹, respectively. The D band is attributed to in-plane vibrations of sp²-bonded carbon atoms with structural defects, whereas the G band corresponds to sp²-bonded carbon atoms with ordered crystalline structures.^31,32 The I_D/I_G ratio was found to be around 1.1 confirming defects in the lattice of the graphitic core (sp³-hybridized carbon networks) and carbon framework (sp²-hybridized carbon networks) after fs laser exposure.^26,33 The fundamental bands for cellulose were also observed at 2948 and 1115 cm⁻¹ which corresponds to C–H stretching and asymmetric stretching vibration of the C–O–C glycosidic linkage, respectively.³⁴ The peaks observed at 921 and 750 cm⁻¹ can be associated with C–H and C–OH groups, respectively.³⁴ The surface functionalization of the patterned substrate was analyzed using XPS. High-resolution scans of C1s and O1s were compared for flat and patterned CA. In the case of the flat substrate (Fig. S3(i)), C1s comprised three peaks at 284.6, 286.4 and 288.8 eV corresponding to sp³ C–C, C=O and O–C=O groups, respectively, which complies with the chemical composition of cellulose.³¹ Fig. 4B(i) shows the C1s peaks for the patterned substrate where 284.6, 285.6, 286.6 and 288.3 eV correspond to sp² C=C, sp³ C–O, C=O and O–C=O functionalities, respectively.³⁵ The appearance of the 285.6 eV peak in the patterned CA indicates the oxidation of the material due to fs laser exposure (Fig. 4B(i)). O1s in the case of flat substrate showed peaks at 532.0 and 533.1 eV for C=O and C–OH (Fig. S3(ii)). For patterned substrate O1s peaks were observed at 531.5 and 532.6 eV representing C=O and C–O groups (Fig. 4B(ii)). The intensity of the C–O group was also found to increase after laser exposure, which was attributed to the enhanced oxidized carbon functionalities after patterning with fs laser exposure.³⁶

Fig. 4 (A) Raman spectra of flat and patterned CA and (B) XPS spectra for patterned CA (i) C1s and (ii) O1s.

3.3. Cytocompatibility and morphological observations

The cytocompatibility of the patterned CA substrate was evaluated using C2C12 cells to explore their potential applications in cell culture. The inherent orientation of muscle cells in vivo emphasizes the significance of cellular organization in maintaining the structure and function of tissues, making C2C12 cells an ideal model system.³⁷ The cell attachment and growth were checked on the patterned substrates with anisotropic microgrooves coated with Collagen I as C2C12 cells did not adhere effectively to the uncoated substrates (Fig. S4). After 24 h of incubation, cells were observed under a microscope for growth and morphological features. Calcein staining confirmed that the cells were viable and healthy, which was attributed to the cytocompatibility and non-toxic nature of the substrate (Fig. 5A). The cells seeded on the patterned substrates exhibited attachment and growth in the direction of the pattern. The cells were elongated and aligned in the direction of the pattern by responding positively to the topography indicating the phenomenon of contact guidance, the tendency of cells to align, migrate, or elongate along topographical features.³⁸ Contact guidance is well reported in various cell types such as hMSCs, fibroblasts, myoblasts and neuronal cells on different patterns such as gratings,³⁹ grooves⁴⁰ or ridges.⁴¹ The SEM micrographs further confirmed the cellular alignment and position of the cells on the patterned substrates (Fig. S5). This demonstrates the potential of patterned substrates to achieve controlled cellular alignment, an important physiological phenomenon observed in embryogenesis, tissue maturation and regeneration.⁸ No typical orientation was observed in the cells cultured on isotropic mesh structures (Fig. S6). Although the cells adhered to and grew on the CA film, their growth was random in orientation (Fig. 5A(i–iii)), emphasizing the crucial role of the pattern on the substrate in guiding cells.

Fig. 5 Response of mouse myoblast cells (C2C12) on flat and patterned substrates: (A) Calcein-Hoechst stained images of C2C12 cells on flat (i–iii) and patterned (iv–vi) substrates (scale bar: 200 μm) with cellular parameters such as (B) area, (C) circularity and (D) aspect ratio.

The morphological features of the cells on the CA-patterned substrates were evaluated by analysing the cellular area, circularity, and aspect ratio using Fiji (ImageJ). The cells showed an average cellular area of 648 ± 200 μm² on the patterned substrate, whereas on CA film the average area was 907 ± 236 μm² (Fig. 5B). The elongated and aligned morphology of the cells was attributed to the ability of the pattern to influence cellular circularity and aspect ratio. The circularity and aspect ratio for cells growing on the patterned substrate were 0.24 ± 0.07 and 6 ± 2, respectively. There was a significant difference when compared with cells on flat CA film where the circularity and aspect ratio were 0.37 ± 0.11 and 3 ± 1, respectively (Fig. 5C and D). These observations are consistent with previously reported studies, where topographical cues have been shown to control the morphological features of adherent cells.^42–44

3.4. Cellular orientation and nuclear morphology

Anisotropic patterned substrates are known to guide the orientation and alter the morphology of different cells, and they are widely used for cells such as C2C12, where the alignment of cells is crucial in achieving physiological relevance. Therefore, the orientation of the cells on anisotropic patterned substrates was investigated by measuring the orientation angle of the nucleus (Fig. 6A). The nucleus is the controlling center of cells, and is known to be influenced by topographical cues that regulate the expression of genes involved in vital cellular function and, hence, fate.⁴⁵ It has also been reported that changes in the size, shape, and orientation of cells are linked to those of the nucleus of cells, which in turn regulates cellular behavior by modulating gene expression.⁴⁶ Here, the Hoechst stained nuclei of cells on the CA film showed a circular shape (Fig. 6B(i)), whereas elongated nuclei (Fig. 6B(ii)) with a high aspect ratio aligned in the direction of the pattern were observed on the patterned CA substrate, showing the influence of the patterned substrate on the nuclear orientation. The results showed that the majority of cells were aligned on a patterned substrate with an orientation angle of less than 20°. Fig. 6C shows the distribution of cells with different orientation angles. For the anisotropic patterned substrate, the average angle of cellular orientation was found to be 5.0 ± 5°. In the case of CA film, the angle of orientation was 39.6 ± 19°, indicating random growth of cells.

Fig. 6 (A) Schematic showing the measurement of cellular orientation on patterned and flat substrates. (B) Hoechst-stained images of nuclei on (i) flat and (ii) patterned substrates (scale bar: 10 μm). (C) Analysis of cellular alignment on flat and patterned substrates and its influence on nuclear (D) area, (E) circularity and (F) aspect ratio.

Furthermore, the effect of CA patterned substrates on cellular nuclei was evaluated by analysing nuclear morphological features such as area, circularity and aspect ratio using Fiji (ImageJ). The cells showed an average nuclear area of 192 ± 68 μm² on the patterned substrate, whereas on CA film the average area was 213 ± 39 μm² (Fig. 6D). The influence on nuclear circularity and aspect ratio was also examined because the elongated and aligned morphology of the cells was linked to the pattern's effect on the cells' circularity and aspect ratio. The results showed that the circularity and aspect ratio for nuclei of cells growing on a patterned substrate were 0.86 ± 0.04 and 1.6 ± 0.2, respectively. There was a significant difference when compared with the nuclei of cells on a flat CA film where the circularity and aspect ratio were 0.95 ± 0.01 and 1.1 ± 0.1, respectively (Fig. 6E and F). The difference in nuclear parameters on the patterned and flat substrates confirms the mechanosensitivity of the nucleus to underlying topographical cues.³

The results emphasize the potential of the CA patterned substrate with micro/nanostructures in regulating cellular and nuclear morphology and controlling cellular alignment. By modulating cellular orientation and potentially directing mechanotransduction pathways, these patterned platforms can be tailored to promote lineage-specific differentiation.⁴⁷ Moreover, the defined patterns may allow for spatial control over cell behavior, such as adhesion, proliferation and orientation enabling an in vivo like microenvironment. This highlights the potential of the patterned substrates with micro/nanostructures in a wide range of applications such as tissue engineering,⁴⁸ drug delivery,⁴⁹ biosensing⁵⁰ and understanding fundamental cell-substrate interactions and mechanobiology.⁵¹

3.5. Analysis of actin expression and orientation

Actin stress fibers, key components of the cytoskeleton, play a crucial role in maintaining cellular polarity, shape, and size.⁵² Topographical features can influence their crosslinking density, length, and anisotropy, thereby impacting cellular behavior. To assess this effect, F-actin expression and actin stress fiber orientation were analyzed using actin staining on patterned substrates (Fig. 7A). Cells cultured on patterned surfaces exhibited higher actin expression (124.6 ± 23.4) than those on flat surfaces (54.8 ± 16.4) (Fig. 7B). Additionally, actin stress fibers were aligned along the direction of the pattern, as confirmed by coherency measurements (Fig. 7C) using the OrientationJ plugin in ImageJ. A coherency value of 1 indicates complete fiber alignment, whereas 0 represents random orientation. The highest coherency (0.22 ± 0.04) was observed in cells on patterned substrates compared to flat controls (0.11 ± 0.07). The directionality of actin stress fibers in a single cell is also shown in Fig. S7. The histogram shows the distribution of stress fibers in the cells on flat and patterned substrates. This suggests that topographic cues from patterned substrates influence stress fiber redistribution and alignment along the pattern direction, thereby regulating cellular shape and size.⁵³ This is mainly due to the mechanosensing machinery of cells where focal adhesions, which serve as anchorage points between the extracellular matrix and the cytoskeleton, tend to form preferentially along topographical features, leading to anisotropic stress fiber formation and nuclear elongation.⁵⁴ This reorganization of stress fibers is closely linked to changes in nuclear morphology, as observed in aligned cells on patterned substrates (Fig. 6B). A potential mechanism underlying this phenomenon involves cytoplasmic actin filaments exerting lateral compressive forces on the nucleus, which in turn modulates nuclear morphology and regulates gene expression in response to changes in cell shape, as reported in previous studies.⁴⁶ Several studies have shown that patterned surfaces can influence key mechanotransduction pathways such as YAP/TAZ, Wnt, Hippo signalling, actin remodelling, and calcium signalling, each of which plays a vital role in regulating gene expression and cellular behavior.^55–57

Fig. 7 (A) Expression of actin in C2C12 cells cultured on patterned and flat cellulose acetate substrates (scale bar: 50 μm), (B) actin intensity and (C) actin coherency.

Conclusion

In conclusion, we report the fabrication of micro/nanostructured patterns on a biocompatible CA to regulate cellular behavior and orientation using fs laser lithography. The fs laser fabrication of CA provides high resolution, precision, reproducibility, cost-effectiveness, contactless processing, versatility, and automation potential. The anisotropic pattern on the cellulose acetate substrate showed cellular growth and alignment attributed towards the potential of this engineered substrate to control the cellular morphology, shape and actin coherency. The patterned substrate also showed altered nuclear morphology confirming the influence of topographical cues, which may be responsible for the expression of different genes involved in adhesion, growth and cytoskeletal organization. These results are particularly important when designing biomaterials to elicit desired cellular responses. Overall, the engineered micro/nanostructured cellulose acetate patterns provide a platform for mimicking the in vivo like microenvironment, which enables fundamental and application-based studies in mechanobiology, tissue engineering and regenerative medicine.

Author contributions

T. S. – data curation and interpretation, formal analysis, writing – original draft, review and editing, T. A. – data curation and interpretation, formal analysis, R. Y. – data curation and interpretation, formal analysis, Su. S. – editing, project administration, resources, funding acquisition, A. M. – supervision, validation, writing review and editing, project administration, resources, funding acquisition, Sh. S. – conceptualization, supervision, validation, writing review and editing, project administration, resources, funding acquisition.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Data availability

The data supporting this article have been included as part of the SI. See DOI: https://doi.org/10.1039/d5tb01122a

Acknowledgements

The authors thank Dr Jyotsna Dhawan's laboratory at the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India for kindly gifting C2C12 cells. All authors acknowledge the support from the Sophisticated Analytical Instrument Facility (SAIF) and Central Facility, Industrial Research and Consultancy Center (IRCC), Indian Institute of Technology Bombay (IITB) for providing characterization facilities. The authors thank Dr Rahul Kumar Das for his valuable suggestions. TS and RY acknowledge the Prime Minister's Research Fellowship, Ministry of Education, Government of India.

References

1. P. Jain, S. B. Rauer, M. Möller and S. Singh, Biomacromolecules, 2022, 23, 3081–3103.
2. H. Atcha, Y. S. Choi, O. Chaudhuri and A. J. Engler, Cell Stem Cell, 2023, 30, 750–765.
3. E. Sarikhani, D. P. Meganathan, A. K. K. Larsen, K. Rahmani, C. T. Tsai, C. H. Lu, A. Marquez-Serrano, L. Sadr, X. Li, M. Dong, F. Santoro, B. Cui, L. H. Klausen and Z. Jahed, ACS Nano, 2024, 18, 19064–19076.
4. M. Ermis, E. Antmen and V. Hasirci, Bioact. Mater., 2018, 3, 355–369.
5. Y. Wang, H. Liu, H. Wang, H. Xie and S. Zhou, J. Mater. Chem. B, 2024, 12, 6690–6702.
6. F. M. Refaaq, X. Chen and S. W. Pang, Sci. Rep., 2020, 10, 1–11.
7. C. Leclech and C. Villard, Front. Bioeng. Biotechnol., 2020, 8, 551505.
8. Y. Li, G. Huang, X. Zhang, L. Wang, Y. Du, T. J. Lu and F. Xu, Biotechnol. Adv., 2014, 32, 347–365.
9. W. Liu, Q. Sun, Z.-L. Zheng, Y.-T. Gao, G.-Y. Zhu, Q. Wei, J.-Z. Xu, Z.-M. Li, C.-S. Zhao, W. Liu, Q. Sun, Z.-L. Zheng, Y.-T. Gao, Q. Wei, J.-Z. Xu, Z.-M. Li, C.-S. Zhao and G.-Y. Zhu, Small, 2022, 18, 2104328.
10. H. Özçelik, C. Padeste and V. Hasirci, Colloids Surf., B, 2014, 119, 71–81.
11. J. I. Kim, T. I. Hwang, L. E. Aguilar, C. H. Park and C. S. Kim, Sci. Rep., 2016, 6, 1–12.
12. C. Acikgoz, M. A. Hempenius, J. Huskens and G. J. Vancso, Eur. Polym. J., 2011, 47, 2033–2052.
13. D. Baek, S. H. Lee, B. H. Jun and S. H. Lee, Adv. Exp. Med. Biol., 2021, 1309, 217–233.
14. Y. Zhang, X. Wang, K. Yan, H. Zhu, B. Wang, B. Zou, Y. Zhang, X. Wang, B. Zou, B. Wang, K. Yan and H. Zhu, Adv. Funct. Mater., 2023, 33, 2211272.
15. D. Zhu, P. Zuo, F. Li, H. Tian, T. Liu, L. Hu, H. Huang, J. Liu and X. Qian, Colloid Interface Sci. Commun., 2024, 59, 100770.
16. Y. Lu, Y. F. Li, G. Wang, Y. Yu, Z. Bai, Y. Wang and Z. W. Lu, Adv. Mater. Technol., 2023, 8, 2300015.
17. S. Ravi Kumar, B. Lies, H. Lyu and H. Qin, Procedia Manuf., 2019, 34, 316–327.
18. B. J. Reichert, S. Brückner, H. Bartelt and K. D. Jandt, Adv. Eng. Mater., 2007, 9, 1104–1113.
19. K. O. Böker, F. Kleinwort, P. Simon, K. Jäckle, S. Taheri, W. Lehmann and A. F. Schilling, Materials, 2020, 1–16.
20. A. M. Alshehri, AIP Adv., 2021, 11, 085017.
21. R. J. Hickey and A. E. Pelling, Front. Bioeng. Biotechnol., 2019, 7, 1–15.
22. H. Seddiqi, E. Oliaei, H. Honarkar, J. Jin, L. C. Geonzon, R. G. Bacabac and J. Klein-Nulend, Cellulose, 2021, 28, 1893–1931.
23. W. Cheng, Y. Zhu, G. Jiang, K. Cao, S. Zeng, W. Chen, D. Zhao and H. Yu, Prog. Mater. Sci., 2023, 138, 101152.
24. R. P. Chaudhary, A. Jaiswal, G. Ummethala, S. R. Hawal, S. Saxena and S. Shukla, Addit. Manuf., 2017, 16, 30–34.
25. S. Rani, R. K. Das, T. Suryawanshi, A. Jaiswal, A. Majumder, W. Cheng, S. Saxena and S. Shukla, Small, 2025, 21, 2405928.
26. F. Morosawa, S. Hayashi and M. Terakawa, ACS Sustain. Chem. Eng., 2021, 9, 2955–2961.
27. P. Samyn, J. Everaerts, A. M. Chandroth, P. Cosemans and O. Malek, Carbohydr. Polym., 2024, 340, 122307.
28. F. Beinhorn, J. Ihlemann, K. Luther and J. Troe, Appl. Phys. A: Mater. Sci. Process., 2004, 79, 869–873.
29. D. Zhang and L. Guan, Laser Ablation, Elsevier, 2014, vol. 4.
30. L. Cui, X. Ren, J. Wang and M. Sun, Mater. Today Nano, 2020, 12, 100091.
31. V. Kuzmenko, N. Wang, M. Haque, O. Naboka, M. Flygare, K. Svensson, P. Gatenholm, J. Liu and P. Enoksson, RSC Adv., 2017, 7, 45968–45977.
32. Y. R. Rhim, D. Zhang, D. H. Fairbrother, K. A. Wepasnick, K. J. Livi, R. J. Bodnar and D. C. Nagle, Carbon, 2010, 48, 1012–1024.
33. Y. R. Kang, Y. L. Li, F. Hou, Y. Y. Wen and D. Su, Nanoscale, 2012, 4, 3248–3253.
34. J. A. Sánchez-Márquez, R. Fuentes-Ramírez, I. Cano-Rodríguez, Z. Gamiño-Arroyo, E. Rubio-Rosas, J. M. Kenny and N. Rescignano, Int. J. Polym. Sci., 2015, 320631.
35. Y. Du, Y. Li and T. Wu, RSC Adv., 2017, 7, 41838–41846.
36. C. Huang, C. Y. Tsai, R. S. Juang and H. C. Kao, J. Appl. Polym. Sci., 2010, 118, 3227–3235.
37. F. Greco, T. Fujie, L. Ricotti, S. Taccola, B. Mazzolai and V. Mattoli, ACS Appl. Mater. Interfaces, 2013, 5, 573–584.
38. G. Thrivikraman, A. Jagie, V. K. Lai, S. L. Johnson, M. Keating and A. Nelson, Proc. Natl. Acad. Sci. U. S. A., 2021, 118, 1–11.
39. L. M. Murray, V. Nock, J. J. Evans and M. M. Alkaisi, J. Biomed. Mater. Res., Part A, 2016, 104, 1638–1645.
40. A. Sharaf, J. P. Frimat and A. Accardo, Mater. Today Bio, 2024, 29, 101325.
41. S. Watari, K. Hayashi, J. A. Wood, P. Russell, P. F. Nealey, C. J. Murphy and D. C. Genetos, Biomaterials, 2012, 33, 128–136.
42. S. Yadav and M. Abhijit, Bioinspir. Biomim., 2022, 17, 056002.
43. C. Wu, C. S. M. Chin, Q. Huang, H. Y. Chan, X. Yu, V. A. L. Roy and W. J. Li, Microsystems Nanoeng., 2021, 7, 1–15.
44. J. Carthew, H. H. Abdelmaksoud, K. J. Cowley, M. Hodgson-Garms, R. Elnathan, J. P. Spatz, J. Brugger, H. Thissen, K. J. Simpson, N. H. Voelcker, J. E. Frith and V. J. Cadarso, Adv. Funct. Mater., 2022, 32, 2100881.
45. K. Anselme, N. Tusamda Wakhloo, P. Rougerie, L. Pieuchot, K. Anselme, N. T. Wakhloo, L. Pieuchot and P. Rougerie, Adv. Healthcare Mater., 2018, 7, 1701154.
46. M. Versaevel, T. Grevesse and S. Gabriele, Nat. Commun., 2012, 3, 1–11.
47. K. Kamguyan, S. Z. Moghaddam, A. Nazbar, S. M. A. Haramshahi, S. Taheri, S. Bonakdar and E. Thormann, Nanoscale Adv., 2021, 3, 333–338.
48. A. K. Gaharwar, I. Singh and A. Khademhosseini, Nat. Rev. Mater., 2020, 5, 686–705.
49. G. He, H.-J. Chen, D. Liu, Y. Feng, C. Yang, T. Hang, J. Wu, Y. Cao and X. Xie, Adv. Mater. Interfaces, 2018, 5, 1701535.
50. S. Fruncillo, X. Su, H. Liu and L. S. Wong, ACS Sens., 2021, 6, 2002–2024.
51. A. V. Singh, R. Patil, D. K. Thombre and W. N. Gade, J. Biomed. Mater. Res., Part A, 2013, 101, 3019–3032.
52. P. S. Mathieu and E. G. Loboa, Tissue Eng., Part B, 2012, 18, 436–444.
53. P. Bhattacharjee, B. L. Cavanagh and M. Ahearne, Colloids Surf., B, 2020, 190, 110971.
54. W. Xie, X. Wei, H. Kang, H. Jiang, Z. Chu, Y. Lin, Y. Hou and Q. Wei, Adv. Sci., 2023, 10, 2204594.
55. Y. A. Miroshnikova, S. Manet, X. Li, S. A. Wickström, E. Faurobert and C. Albiges-Rizo, Mol. Biol. Cell, 2021, 32, 1724.
56. R. Cao, H. Tian, Y. Tian and X. Fu, Adv. Sci., 2024, 11, 2302327.
57. J. M. Stukel and R. K. Willits, Tissue Eng., Part B, 2016, 22, 173–182.

---