Decellularized grass as a sustainable scaffold for skeletal muscle tissue engineering

Scaffold materials suitable for the scale-up and subsequent commercialization of tissue engineered products should ideally be cost effective and accessible. For the in vitro culture of certain adherent cells, synthetic fabrication techniques are often employed to produce micro- or nano-patterned substrates to influence cell attachment, morphology, and alignment via the mechanism of contact guidance. Here we present a natural scaffold, in the form of decellularized amenity grass, which retains its natural striated topography and supports the attachment, proliferation, alignment and differentiation of murine C2C12 myoblasts, without the need for additional functionalization. This presents an inexpensive, sustainable scaffold material and structure for tissue engineering applications capable of influencing cell alignment, a desired property for the culture of skeletal muscle and other anisotropic tissues.

For applications in regenerative medicine, the use of animal tissue presents the advantage of structural similarity and even like-for-like organs, but the main disadvantage is that they still require donor tissues or organs, introducing ethical and/or availability concerns. The abundance of plants in nature means their decellularized readyformed structures present a sustainable, ethical and inexpensive input alternative. Plant tissues that have been decellularized by detergent treatment and re-seeded with mammalian cells include apple slices, spinach leaves, bamboo, orchid, vanilla, anthurium magnificum and anthurium warocqueanum, parsley, calathea zebrina, wasabi, green onion, celery, carrot, broccoli, sweet pepper, persimmon and jujube. 8,9,[13][14][15][16][17] The cell wall in plants, as a type of ECM, remains intact and is primarily cellulose, which is the most abundant organic polymer on earth 18 and has been shown to be cytocompatible in various studies. 8,9,13,[19][20][21][22][23] Tissue engineering of skeletal muscle has been reviewed for potential applications in regenerative medicine, tissue replacement, cell-based therapies, drug testing and toxicology screening, and for consumption in the form of cultured meat, also known as in vitro and cell-based meat. 3,[24][25][26][27][28] The final form and functionality of the engineered tissue will differ depending on the intended application, however engineering skeletal muscle in vitro with the intent of achieving functional muscle requires uniaxial alignment of muscle fibres due to the directional-dependent, anisotropic characteristic and structure of muscle. Studies have shown that alignment of myoblasts prior to differentiation enables aligned myotubes to be engineered. 29,30 Methods of such alignment include active stimulation (electrical or mechanical) or passive methods such as modifying substrate mechanical properties, for example, stiffness, or surface morphology to introduce topographic cues that promote cell alignment via contact guidance. 23,[31][32][33][34][35][36][37][38] Grass as a proposed scaffold material meets the criteria of being readily available due to its abundance in nature and it was hypothesized that the long narrow leaves and parallel vasculature system in grass blades, a characteristic of monocot plant leaves, may support the self-alignment of cells following seeding. Hence, the aim of this study was to investigate the novel prospect of using decellularized grass as a scaffold for the in vitro culture of myoblasts. Here we report the decellularization of amenity grass, the characterization of its topography and its cytocompatibility with murine myoblasts in terms of attachment, viability, proliferation, and differentiation. Overall, this study demonstrates the suitability of decellularized grass as a scaffold for the tissue engineering of aligned myofibres via the passive mechanism of contact guidance.

| Grass decellularization
Grass blades were obtained from the University of Bath grounds. This is amenity grass suspected to be a combination of rye grass, fescue grass, and/or annual bluegrass. Blades were pre-treated by washing for 5 min with ethanol (VWR Chemicals, 20821) followed by 5 min with phosphate-buffered saline (PBS; Sigma-Aldrich, D8537). This sequential washing was repeated a further two times. Grass blades were then soaked and agitated in 1% (w/v) sodium dodecyl sulphate (SDS; Sigma-Aldrich, L4509), 1% (v/v) Tween-20 (Sigma-Aldrich, P1379), and 10% (v/v) bleach (The Consortium, 049403) at 150 ml decellularization working solution per gram of grass for 1 to 2 days until all blades were visually translucent. Following decellularization, grass blades were washed with MilliQ water and stored in PBS at 4 C prior to use.

| Measurement of grass blade thickness
The thickness of native and decellularized grass was measured using an electronic micrometer (RS Pro External Micrometer, 705-1,229, range 0-25 mm, resolution 0.001 mm). Data are expressed as the mean ± SD of the reported sample size of n individual grass blades using the average of two readings at different points along the length of each blade. Measurements were conducted on grass blades air dried for >3 hr at room temperature. Native grass was cut fresh prior to drying out. Decellularized grass was removed from 4 C PBS prior to drying.

| Scanning electron microscopy
Grass blades, either pre-or post-decellularization, were frozen at À80 C for 3 hr and lyophilized overnight using a Thermo Savant MicroModulyo-230 freeze dryer. Samples were then coated with a 50 nm layer of gold in an Edwards Sputter Coater S150B and imaged on a JEOL JSM-6480LV SEM.

| Atomic force microscopy
Atomic force microscopy (AFM) was utilized to determine the topography of lyophilized samples of decellularized grass in ambient air.
AFM experiments were performed in tapping mode on a Bruker Multimode Nanoscope IIIA AFM machine with a NuNano Scout 70 silicon probe (spring constant of 2 N/m and resonant frequency of 70 kHz). Gwyddion software was used to analyse the AFM images and generate the overlay 3D images of the topography.

| Profilometry
A Proscan 2000 non-contact surface profilometer with a chromatic sensor (sensor type S5/03) at a sample rate of 300 Hz and 200 steps (step size of 2 μm) was used to characterize the surface morphology of lyophilized samples of decellularized grass in ambient air, in the micrometre range.

| Cell culture
The murine myoblast cell line C2C12 (ECACC 91031101) was used to model skeletal muscle cells. Cells were maintained in proliferation medium consisting of high glucose Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich D5796) supplemented with 10% (v/v) foetal bovine serum (FBS; Gibco™, Thermo Fisher Scientific 10270106) and 1% (v/v) penicillin/streptomycin (P/S; Sigma-Aldrich P4333) in a humidified incubator at 37 C and 5% CO 2 . Cells were maintained in T-75 culture flasks and passaged approximately every 3 days until reaching $80% confluence, at which point they were sub-cultured.

| Cell adhesion on decellularized grass
Prior to cell seeding, grass blades were sterilized by soaking in 70% (v/v) ethanol (VWR Chemicals, 20821) for 1 hr, followed by washing in proliferation medium and pre-treatment by soaking in proliferation medium overnight at 4 C. Attachment of C2C12 cells to native grass (i.e., blades incubated in 70% ethanol for 1 hr but, otherwise, untreated) or decellularized grass was then assessed by seeding a 1 cm long section of an untethered grass blade with 5,000 cells cm À2 , relative to the surface area of a well, in a 24-well cell culture plate. Cells were seeded by pipetting 0.

| Differentiation of myoblasts on decellularized grass
Differentiation of C2C12 cells on decellularized grass was assessed by seeding 1 cm long sections of untethered grass blades with 50,000 cells cm À2 , relative to the surface area of a well, in a low-attachment 24-well plate. Cells were cultured in proliferation medium for 2 days and then differentiation medium for 7 days. Differentiation medium consisted of high glucose DMEM supplemented with 2% (v/v) horse serum (Sigma-Aldrich, H1270) and 1% (v/v) P/S. Samples were then stained using FDA and Hoechst and imaged with a Leica DMI4000B microscope as described above.

| Directionality and cell orientation analysis
Cell alignment on decellularized grass and tissue culture plastic was assessed using 10Â magnification images obtained from the proliferation assay described above. Fluorescent images of C2C12 cells stained with FDA on decellularized grass were compared with brightfield images of C2C12 cells in multiwell tissue culture plastic plates, both seeded at 5,000 cells cm À2 based on the surface area of a well. Images were converted to 8-bit and cropped to the in-focus portion of the image for the grass blades. The angle of the grass blade was measured using the ImageJ line measurement tool and the image then rotated with a reference point of 0 in the East direction (degree of rotation differed per image based on the angle of grass in the raw image, with images rotated so that grass blades were horizontal). Cell alignment was quantified using the Directionality plug-in of the image analysis software suite, Fiji (a distribution of ImageJ) 39 with the Fourier components method (number of bins = 90, between À90 and 90 ). The same method was used to quantify the alignment of multinuclear myotubes.
Cell alignment on decellularized grass was also assessed based on orientation of nuclei using the image processing software, Cel-lProfiler™. 40 A pipeline was developed to identify and measure the orientation of Hoechst-stained nuclei in 20Â magnification images from the proliferation assay as described above. Nuclei were identified in grey-scale images using the IdentifyPrimaryObjects module, with the following advanced settings: 40 to 100 pixel object diameter,

| Statistical analysis
Captions for figures and tables describing experimental results state the number of experimental repeats (n), replicates (N) and error.
Results are presented as mean ± standard deviation (SD) unless otherwise stated. A type two (unpaired) two-tailed t test, using the Analysis ToolPak add-in for Microsoft Excel, was used to assess significant differences between groups, comparing means of independent biological replicates unless otherwise stated. A value of p < 0.05 was considered statistically significant.

| Decellularization of grass
The applicability of decellularized grass as a scaffold for the culture of mammalian cells required confirmation that the decellularization process employed was sufficient to remove the native plant cells and genetic material, while maintaining the ECM structure, to prevent cytocompatibility issues in vitro and potential adverse host response in vivo. 11 To this end, the degree of grass decellularization was F I G U R E 1 (a,b) Images of native grass (a) and decellularized grass (b). Scale bars: 1 cm. (c-f) Hoechst 33342 staining (blue) for nuclei in native grass (c,e) and decellularized grass (d,f). Scale bars: 200 μm (c,d) and 100 μm (e,f) F I G U R E 2 (a to f) 3D AFM images of decellularized grass and the corresponding topographic height plots (g) normalized to the minimum zaxis data point. Arrows = longitudinal direction of grass blade. Black dotted line = specific cross-section mapped onto the height plot determined by visual inspection, using a colour and transparency sight-test (Figure 1(a,b)). When the grass blades had been stripped of all colour and become ''ghost-like'', decellularization was deemed complete. Following the decellularization process, the thickness of the grass blades decreased from 81 ± 11 μm (n = 18) for native grass to 75 ± 11 μm (n = 18) for decellularized blades (p > 0.05). Further qualitative confirmation of decellularization was conducted by Hoechst staining for nuclei, as one of the cell removal verification methods, 6 illustrating the absence of nuclei following the decellularization process ( Figure 1(c-f)).

| Topography of decellularized grass
The hypothesized benefit of using grass as a plant-source scaffold is the anisotropic form of parallel vasculature presenting a natural surface pattern that may present cues for the passive alignment of cells. This presented the need to characterize the surface topography of grass to determine whether it was affected by the decellularization process. In

| Cell attachment, proliferation and viability on decellularized grass
The use of a material as a cell scaffold is dependent on its cytocompatibility and consequent ability to support the attachment and proliferation of cells. With decellularized grass scaffolds in the context of this report, this represents the ability of the remaining plant ECM to support cell adhesion and growth. Without functionalization, decellularized grass supported the attachment of C2C12 cells following a 3-hour attachment period with greater attachment efficiency than on native grass (Figure 4(a)). The attachment of C2C12 cells in F I G U R E 3 Decellularized grass blade surface morphology and topography visualized using a non-contact profilometer scan of a 400 Â 400 μm mapping area this case was not comparable to tissue culture plastic (TCP), as the grass blades were not clamped or fixed to the bottom of the well. As a result, the percentage of cells contacting the available grass surface area was less than the number that would contact the plastic in a normal well. Figure 4

| Quantification of cell alignment
To determine the effect of topographical cues presented by the natural surface topography of grass on cell behaviour, the orientation of present on the blades. This was quantified as 43% ± 8% (θ < ± 10 ) and 59% ± 8% (θ < ± 20 ), increasing up to 82% ± 5% (θ < ± 50 ) from quantitation via the Directionality plug-in for ImageJ (n = 3, mean ± SD). While the percentage of cells with a preferred orientation on TCP of 14% ± 2% (θ < ± 10 ) can be classified as random.

| Myoblast differentiation on decellularized grass
A vital feature of a biomaterial scaffold for engineered skeletal muscle is its ability to support the differentiation of mononuclear cells such as myoblasts or satellite cells into myotubes, ideally in an aligned manner mimicking the anisotropic structure of natural muscle. Differentiation of C2C12 myoblasts on decellularized grass was confirmed following the culture of confluent cells in differentiation medium with a reduced serum content; Figure 7 clearly indicates the presence of aligned, multinuclear myotubes on decellularized grass scaffolds. Alignment of myotubes was quantified using the ImageJ Directionality plug-in (Figure 7(b)).

| DISCUSSION
This article describes the use of decellularized grass scaffolds for the culture of C2C12 myoblasts, a model cell line for skeletal muscle tissue engineering applications. The detergent-based decellularization process was adapted from prior studies on plants 8,9,13 and is a simple procedure that can be performed in-house with the final-form scaffolds obtained in 1 to 2 days (Figure 1). With one of the potential applications of engineered muscle being for human consumption as cultured meat, our use of ethanol in the pre-treatment phase and Tween-20 as the non-ionic detergent, are favourable over the previously reported use of hexane and Triton X-100, respectively. 7,8,13 However, it is likely that traces of hexane and Triton X-100 would be removed following washing, sterilization and pre-treatment steps before cell seeding, as highlighted by Adamski, et al, through cell cytocompatibility studies. 42 Furthermore, for the application of edible scaffolds for cultured meat, it may not be sufficient for the substrate to be edible, non-toxic and texture-enhancing, but also nutritionally beneficial. In the case of decellularized grass, the complex cell walls of most grass species (save anomalies like maize) are not typically digestible without rumination. The composition of grass cell walls, as monocots, differ from most dicots, with greater proportions of hemicelluloses in both cell walls and lignin and silica in the secondary cell wall. 10 Consequently, further nutritional, compositional and bioavailability analysis should be conducted on decellularized grass preand post-cooking. In addition, the texture of food has a significant influence of consumer acceptance, and this will also require further investigation for the adoption of plant-based scaffolds in clean meat applications.
Decellularized grass as a naturally derived scaffold retains a topography comprised of parallel grating-like grooves. AFM and profilometry scans of decellularized blades (Figures 2 and 3), demonstrated that the surface topography of the grass blades consisted of longitudinal, parallel striations, in the uniaxial direction, a feature also visible using light microscopy ( Figure S2). Topography analysis indicated the presence of grooves at both the nano-and micro-scale with variability resulting from the natural source (nano-scale in Figure 2 plots A, B and D to F (AFM), micro-scale in Figure 2 29,34,35,41,[43][44][45] The applicability of decellularized grass as a scaffold for skeletal muscle tissue engineering was assessed through the in vitro culture of C2C12 cells. The results demonstrated that decellularized grass supported the culture of C2C12 cells without the need for functionalization or modification, as demonstrated by the attachment efficiency of 35% ± 7%, significantly greater than the 9% ± 7% for native grass (Figure 4(a)). This is suspected to be a result of removing the waxy cuticle and exposing the ECM of the decellularized grass. The relatively low attachment efficiency of 35% is likely to be the result of the seeding method, where grass blades were untethered in the well, settling on the bottom of the well due to their density, rather than being tethered in any way. Attachment relies on the probability of passive cell-to-grass contact and this would have been diminished by the untethered scaffold. Nonetheless, these scaffolds displayed a significant level of inherent adhesion despite the lack of modification or optimization of the seeding conditions. Cell proliferation is presented in Figure 4 47 However, as expected, growth was slower than on TCP (19.7 ± 0.5 hr). 48 A high cell viability of $95% over a 7 day proliferation period was demonstrated as another measure of cytocompatibility, indicating that the scaffold is non-cytotoxic in accordance with the ISO standard requirements of viability >70%. 49 This is also comparable with other in vitro studies on decellularized scaffolds such as 98 ± 1% for C2C12 cells on decellularized apple slices after 12 weeks and >95% for cardiac cells on a decellularized heart after 8 days. 7,9 T A B L E 1 Cell alignment on natural and synthetic topographic features. Unless otherwise specified, the cell type is C2C12 myoblasts  Hence, decellularized grass was able to support the adhesion and proliferation of a highly-viable population of C2C12 myoblasts without the need to functionalize the scaffold following the decellularization process, unlike a number of decellularized plant studies which involved treatments such as RGDOPA functionalization, biomineralization, fibronectin coating, crosslinking with glutaraldehyde or modifying surface charge to improve cell attachment on cellulose-based scaffolds. 8,9,13,20 In some cases, such as the apple derived scaffolds used by Modulevsky et al, C2C12 cells were found to proliferate well in both unmodified and modified scaffolds. 9 Previous studies have shown that materials with patterned topography in both the nano-and micrometre scale can influence cell behaviour, and that grooved surfaces (also referred to as grating, channels, ridges, steps and cliffs) support cell alignment along the axis of the grooves. 34,35 The use of decellularized plant tissue, specifically grass as a nano-and micropatterned scaffold, presents the advantages of being significantly cheaper and more readily accessible or available than typical micropattern fabrication techniques, which can be timeconsuming, expensive and require access to intricate equipment. 34 The natural topography of decellularized grass was shown here to support the self-alignment of cells via contact guidance with features in both the nano-and micro-scale, demonstrated by the images and preferred orientation plot in Figure 6. Alignment of C2C12 myoblasts was quantified to evaluate the effect of decellularized grass topographic features on inducing contact guidance driven cell behaviour. Compared to the random orientation of cells on the control of TCP (14% ± 2% within θ < ± 10 ), cell alignment was clearly seen on decellularized grass (43% ± 8% within θ < ± 10 ) as quantified via the Directionality plug-in for ImageJ, satisfying the hypothesis of this study.
Qualitatively, the alignment visualized using images of stained live cells (Figures 4(c) and 6) suggested that the cells were more aligned than suggested by the Directionality plug-in analysis. To further investigate C2C12 cell alignment, analysis of stained nuclei was undertaken using CellProfiler™ to quantify their orientation angle and the results obtained indicated a greater proportion of aligned cells. The alignment results in this study for cells aligned within θ < ± 20 were 59% ± 8% using the Directionality plug-in for ImageJ and 72% ± 1% using Cel-  13 In this study, using the same methodology to determine alignment, 72% ± 1% of cells were within θ < ± 20 and 53% ± 4% were within θ < ± 10 , clearly demonstrating that the microstructures within decellularized grass scaffolds were extremely effective at promoting aligned cell growth. With the goal of engineering skeletal muscle, differentiation of mononuclear cells into multinuclear myotubes is required. Importantly, the fact that decellularized grass is shown to support the alignment of C2C12 myoblasts in a preferred orientation, parallel to the axis of the grass blade, satisfies the prerequisite for passive differentiation into aligned myotubes. 29,30 This was confirmed with C2C12 myoblasts, which fused together on decellularized grass blades to form aligned multinuclear myotubes with lengths of >400 μm (Figure 7), replicating the anisotropic nature of skeletal muscle. While C2C12 cells, being murine myoblasts, are not the intended cells for the generation of engineered skeletal muscle tissue for regenerative medicine or cultured meat, this work presents a model scaffold with applicable features for the culture of skeletal muscle cells for these eventual applications. Cell alignment as a desired feature for tissue engineering, is not solely applicable to skeletal muscle tissue engineering; multiple reviews summarize studies used to guide alignment of endothelial cells, smooth muscle cells, fibroblasts, epithelial cells, corneal endothelial cells, neuroblastoma cells and mesenchymal stem cells directed to neuronal lineages. 33,34,37 Due to the mechanism of contact guidance, the natural topography of decellularized grass has the potential to be applicable to multiple tissue targets for passive alignment via topographic cues.

| CONCLUSIONS
Here, we have demonstrated the novel concept of using decellularized, garden-variety grass as a potential scaffold for skeletal muscle tissue engineering. The results obtained reveal the successful decellularization of grass and its subsequent use as a scaffold that supports the attachment, maintenance of viability, proliferation, and differentiation of C2C12 cells. The attachment results indicate C2C12 cells preferentially adhere to grass that has been decellularized over native grass. Topography analysis of grass illustrates how the natural striations, visible by the naked eye, are channel-like, running parallel to the length of grass blades with depth variation in both the nano-and micro-scale. The channelled topography is subject to natural variation and nonhomogeneity but supports the uniaxial self-alignment of C2C12 cells in their myoblast state via contact guidance, a feature retained following differentiation into myotubes. This presents advantages for future skeletal muscle tissue engineering applications as an inexpensive, readily available, preformed scaffold architecture with potential application as a passive means of culturing anisotropic muscle tissue in vitro without the need for additional manipulation to induce cellular alignment. Chris Bowen for access to and use of his profilometer.

Scott Allan and Paul De Bank have no conflicts of interest. Marianne
Ellis is co-founder of Cellular Agriculture Ltd.

DATA AVAILABILITY STATEMENT
The raw and processed quantitative data required to reproduce these findings are available to download from http://dx.doi.org/10.17632/ 5mgnz3zrmv.1