Large and Small assembly: Combining functional macromolecules

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Large and Small assembly: Combining functional macromolecules with small peptides to control the morphology of skeletal muscle progenitor cells Rui Li, Natasha L. Mcrae, Daniel R. McCulloch, Mitchell Boyd-Moss, Colin J. Barrow, David R. Nisbet, Nicole Stupka, and Richard J. Williams Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01632 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Biomacromolecules

Large and Small assembly: Combining functional macromolecules with small peptides to control the morphology of skeletal muscle progenitor cells Rui Li, Natasha L. McRae, Daniel R. McCulloch, Mitchell Boyd-Moss, Colin J. Barrow, David R. Nisbet, Nicole Stupka‡ and Richard J. Williams‡*

AUTHOR ADDRESS Dr. R. Li Centre for Chemistry and Biotechnology, Deakin University, Waurn Ponds, 3216, Australia; Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wenchang, Hainan, 571339, China Prof. C. J. Barrow Centre for Chemistry and Biotechnology, Deakin University, Waurn Ponds, 3216, Australia N. L. McRae, Dr. D. R. McCulloch, Dr. N. Stupka

ACS Paragon Plus Environment

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Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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School of Medicine, Centre for Molecular and Medical Research SRC, Deakin University, Waurn Ponds, 3216, Australia A/Prof. D. R. Nisbet Research School of Engineering, The Australian National University, Canberra, 2601, Australia Biofab3D, St. Vincent’s Hospital, Fitzroy, 3065, Australia Dr. R. J. Williams, Mr. Mitchell Boyd-Moss School of Engineering, RMIT University, Bundoora, 3083, Australia Biofab3D, St. Vincent’s Hospital, Fitzroy, 3065, Australia

KEYWORDS self-assembled hydrogel; functionalisation; peptide; versican; provisional matrix; Tissue Engineering

ABSTRACT The material properties of natural tissues, such as skeletal muscle, are highly sophisticated and are synthetically challenging to mimic. Using natural biomacromolecules to functionalise self-assembled peptide (SAP) hydrogels has the potential to increase the utility of these materials by more closely reproducing the natural cellular environment. Here, to demonstrate that a conserved co-assembly pathway can retain distinct function, the biocompatible peptide derivative Fmoc-FRGDF was co-assembled with either a sulphated polysaccharide, fucoidan, or the provisional matrix proteoglycan, versican. Our results demonstrate

that

thermodynamically

driven

co-assembly

with

biologically

active


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macromolecules is facile, stable, and does not affect the final assembled nanostructure. Biologically, the incorporation of these functionally distinct molecules had no effect on C2C12 myoblast proliferation and viability, but strongly altered their morphology. The surface area of myoblasts cultured on the fucoidan scaffold was reduced at 24 h and 72 h post seeding, with a reduction in the formation of multinucleated syncytia. Myoblasts cultured on versican scaffolds were smaller compared to cells grown on the empty vector scaffolds at 24 h, but not 72 h post seeding, with multinucleated syncytia formation being unaffected. This work allows programmed and distinct morphological effects of cell behaviour, paving the way for further mechanistic studies.

Introduction The extracellular matrix (ECM) is a complex, information-rich structure which is composed of tissue specific matrix macromolecules; such as collagen, laminin, fibronectin and various proteoglycans. These macromolecules work synergistically to present three dimensional (3D) mechanical and chemical signals to cells which regulate all aspects of cell behaviour; including proliferation, adhesion, migration, differentiation and cell death 1. As stem cell technology for regenerative medicine becomes increasingly prevalent, there is a need to develop more advanced biomaterials which better model the three dimensional (3D) cell niche 2. However, controlling stem cells in vitro such that expansion is adequate and the desired phenotype is maintained remains a significant challenge 3. To date, most cell culture approaches use biomacromolecules in two dimensional culture conditions (2D). Therefore, it will be of substantial benefit to reproduce these approaches in materials that more adequately model the spatial and mechanical


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complexities of the natural ECM. Natural materials such as collagen and fibrinogen are highly biomimetic, yet can carry significant risk of pathogen transmission and present immunogenic concerns 4. Alternatively, scaffolds formed from synthetic polymers are easily synthesised and immunocompatible, yet may demonstrate poor degradation under physiological conditions, may require processing using extensive purification steps, and may present concerns regarding the use of toxic chemicals during synthesis.5 A key alternative is to exploit the self assembly of molecules6, and control their formation into organised nanostructures7-8. Low molecular weight hydrogels are formed when small molecules undergo supramolecular self-assembly to entrap their surrounding solvent9. This allows the formation of metastable structures determined by kinetic10 and thermodynamic factors11 driven by dynamic noncovalent intermolecular forces12-13.

Simple 9-

fluorenylmethyloxycarbonyl capped self-assembled peptides (Fmoc-SAPs), first described by Vegners et al. in 199514 for biomaterial applications, offer an alternative approach. These peptides are advantageous as they are readily synthesised, immunocompatible, non-toxic, and effectively degraded in vivo

15

. This approach was further developed by the Xu and

Ulijn research groups. Notably, Xu pioneered a pH controlled trigger for the formation of these peptide scaffolds, allowing them to form under physiological conditions16. Since then, efforts have been made to develop the materials toward attachment based and injectable hydrogels11. These peptides have been functionalised to mimic the attachment signals of the highly expressed ECM glycoprotein fibronectin using the minimalist amino acid sequence arginine-glycine-aspartic acid (RGD), notably by the inclusion of epitopes by the groups of Ulijn17 and

Hamley18. These signals play a significant role in cell adhesion, growth,

proliferation, differentiation and migration

19

. Inspired by this pioneering work, here we


4

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used a previously reported, well characterised, minimalist aromatic pentapeptide, FmocFRGDF, which provides a stable nanofibrous network20 while acting as a carrier for these biomacromolecules. We based this material upon the assembly motif Fmoc-FF

21

which

undergoes self-assembly to form a material via a kinetically entrapped state. However, this material is not optimal as a biomaterial. It has been shown to be unstable and unfunctionalised with some hydrogels degrading within 48 hours leading to cell deattachment and death22. It has been suggested that other peptide sequences can be stable in assembly23.

Notably, a library of longer peptide sequences has been shown to be

thermodynamically stable using a homogeneous sequence and an enzyme enabled reversed hydrolysis reaction24. Therefore, in order to kinetically entrap a longer sequence required for functionalization, we inserted the RGD amino acid sequence between the aromatic residues 20

. We have previously confirmed that this forms a stable, optically transparent hydrogel

where only the Fmoc- and first F are unavailable, with ~20% of the total RGD motifs presented

25

. Indeed we have shown that the Fmoc-FRGDF hydrogel is highly bioavailable

for attachment based cell-culture

20, 26

and biocompatible for in vivo applications

27-28

. We

have recently demonstrated the unique potential of these materials in vivo for brain therapies; specifically to control astrocyte scarring29, deliver and integrate stem cells30 and control the presentation of growth factors31. However, this is only a simple aspect of the ECM environment, and recent efforts have been to utilise multicomponent assembly to enhance the function of the SAP scaffolds32. Indeed, SAPs have been shown to co-assemble with various biomacromolecules33 to form hybrid materials of increased complexity. For example, with structurally and biologically relevant polysaccharides34, polymers35, proteins36 and functional nanomaterials9, where specific


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nanoengineered interactions (e.g., electrostatic, aromatic stacking) and templating are 37

employed to direct the assembly process

. However, the assembly process needs to be

carefully controlled to retain the desirable properties of the peptide nanostructures, as these can be altered dramatically through the co-assembly process36, 38, the type of stimulus used39 and rate at which assembly takes place40-42. Here, we report on the biological effects of two highly sulphated biomacromolecules to demonstrate that the same assembly approach can impart two different cellular environments; a seaweed derived polysaccharide fucoidan, and the developmentally important, proteoglycan versican. These were chosen as they possess similar bulk macromolecular properties (i.e. highly sulphonated, charged) that control the molecular assembly, yet have distinct and separate biological function. Fucoidan is a sulphated polysaccharide which can alter cell adhesion to fibronectin

43

. Whilst fucoidan does have distinct biological effects on various cell lines,

including tissue progenitor cells [21], it is not a component of the natural stem cell niche. Versican is a large chondroitin sulphate proteoglycan which is highly expressed during development and tissue regeneration. Natively, versican is synthesised as part of a provisional matrix that regulates numerous aspects of stem cell behaviour deposition

44

44

, and functions as a scaffold for mature ECM

. Through interactions with various ECM components, versican influences cell

adhesion, proliferation, migration, differentiation and viability

45-46

; however cell behaviour is

not only modulated by versican synthesis, but also by the remodelling and clearance of versican by specific ECM proteases.

45

. Therefore, to ensure that the versican used underwent correct

post-translational modification, and that our approach modelled the established cell culture protocols as effectively as possible, we used a versican formed in conditioned media, where the post-translational addition of chondroitin sulphate side-chains ensures correct biological


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function. This has the advantage of being a well-studied source of biologically relevant versican47-48, and would ensure that the surface properties would be valid for the driving forces involved in self-assembly11. The inclusion of the biomacromolecules is achieved by making them available in solution during the peptide assembly process, using a range of intermolecular forces available through the surface interactions of the fibrillar structures including physical entrapment

49

, hydrophobic

50

and electrostatic interactions 27. This process is sufficiently robust that the fibril nucleation and growth is stable and conserved, yet the biomacromolecues are tightly adsorbed to the surface of the fibrils25 in a supramolecular association, driven by complementary interactions between the structures. There is no covalent bond formation in this system, and therefore no catalyst or cross linking agent which could impinge on cell viability 24. Importantly, the incorporation of a large molecules to the assembly can be tuned so as to not disrupt the ordering of the network, retaining the peptide fibrillar structure51. C2C12 myoblasts were used to investigate the effects of these co-assembled hydrogels on cell viability, proliferation, adhesion and spreading, as well as the formation of multinucleated syncytia. C2C12 myoblasts are a well characterised murine skeletal muscle progenitor cell line used to study cellular processes relevant to myogenesis during skeletal muscle regeneration and development

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. It has been well established in literature that both fibronectin and versican are

important components of the skeletal muscle stem cell niche

53

, and that both versican

45

and

fucoidan 54 can modulate myogenesis in a 2D cell culture environment. Our results show that the Fmoc-SAP scaffolds can be tuned to have specific biological effects on C2C12 myoblasts


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through the stable inclusion of bioactive macromolecules, providing the promise for the development of more complex, biologically active scaffolds for advanced cell culture of tissue progenitor cells. Materials and Methods Solid phase peptide synthesis (SPPS) Fmoc-FRGDF was synthesised using a standard Fmoc chemistry procedure as previously described

55

on a 1.5 mmol scale. The Fmoc-Phe-Wang resin, Fmoc protected amino acids, 2-

(1H-Benzotriazole-1-yl)-1,1,3,3-Tetramethyluronium

hexafluorophosphate

(HBTU),

1-

hydroxybenzotriazole (HOBt), and N,N-diisopropylethylamine (DIPEA) were purchased from GL Biochem. All the other reagents were purchased from Sigma-Aldrich. Reverse phase high performance liquid chromatography (RP-HPLC) was used to assess the purity of the desired sample. Formation of hydrogels All solutions used to make hydrogels for cell culture were sterilised by filtration using a Corning bottle-top vacuum filter systems with a filter size at 0.22 µm (Life Technologies), whereupon assembly was triggered under aseptic conditions. Briefly, 10 mg of Fmoc-FRGDF powder was weighed into a 4 ml glass vial. Then, the peptide was suspended with 400 µL Milli-Q water, and then dissolved by the addition of a minimal volume of 0.5 M NaOH while vortexing, until a transparent solution was obtained. The solution was neutralised by the dropwise addition of 0.1 M HCl to a final pH of 7.4. Immediately following this, growth media, 25 mM glucose Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), was added


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into the solution to bring the total volume up to 1.0 mL. The samples were sealed and left to undergo gelation in a laminar flow cabinet for one hour. Four groups of hydrogels were prepared. (1) 10.0 mg of Fmoc-FRGDF; (2) 10.0 mg of Fmoc-FRGDF along with 0.5 mg of low molecular weight fucoidan (Marinova Pty. Ltd.); (3) 10.0 mg of Fmoc-FRGDF self-assembled with versican enriched conditioned media and (4) 10.0 mg of Fmoc-FRGDF self-assembled with empty vector (pcDNA3.1MycHisA, Life Technologies) conditioned media, as a control for the third group of hydrogels. For each sample in groups 1, 3, and 4, 10.0 mg of Fmoc-FRGDF were placed into 4 mL glass vials. For group 2, 10.0 mg of Fmoc-FRGDF was placed along with 0.5 mg fucoidan. For groups 3 and 4, ¾ volume of growth media, and ¼ volume of versican conditioned media or empty vector conditioned media were used with the volume of FBS adjusted to a final concentration of 10%. To minimise variability, a single batch of versican or empty vector conditioned media was used for all biological and functional experiments. The concentration of fucoidan was determined based on the minimum required to have a biological effect without altering the morphology of the hydrogel significantly 25. The ratio of conditioned media to growth media was based on 2D experiments investigating versican remodelling and myogenesis in C2C12 myoblast 45. Finally, 400 µL of hydrogel from each group was transferred using a 1 ml Pipette into a 24-well cell plate (BD Biosciences) before leaving the hydrogel for 30 min to stabilise, during which time the plates were exposed to under UV light to sterilise the materials prior to seeding the cells

26

. The minimum time required to have a biological effect

without altering the morphology of the hydrogel significantly

25

. The ratio of conditioned to

growth media was based on 2D experiments investigating versican remodelling and myogenesis in C2C12 myoblasts56 Circular dichroism spectroscopy


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Hydrogel spectra were measured using a Jasco J-815 circular dichroism spectrometer with the bandwidth 1 nm and integrations 2 s-1. A 1 mm quartz cell (Starna Pty. Ltd.) was used for all measurements. Samples were prepared as described previously. To avoid overloading the measurement detector, a gel suspension was formed by gentle vortexing to a final peptide hydrogel concentration of 0.4 mg/mL. Data was collected 3 times for each sample and average values were used. The quartz cell was rotated between each measurement to avoid any possible artefacts from linear dichroism. Fourier transform infrared spectroscopy A Nicolet 6700 Fourier transform infrared spectroscopy (FTIR) (ThermoFisher Scientific) was used to collect spectra using attenuated total reflection (ATR) mode. Hydrogels (12 µL) were applied directly to the ATR crystal and scanned between the wave numbers of 4000 and 400 cm-1 over 64 scans. A background scan of DMEM was applied prior to testing the samples. A baseline absorbance of water was subtracted from the total absorbance of the Fmoc-SAPs to suppress interference from the solvent29 Rheometry Using a Discovery Hybrid Rheometer (TA Instruments),a Stress/Strain sweep was performed in order to determine the optimal conditions for the fixed strain frequency sweep. This was done to ensure that the measurements were performed within the linear viscoelastic region. Based on this, the experiments were carried out at constant stress with a strain of 1.0%. An amplitude sweep was performed and showed no variation in G’ and G” up to a strain of 60%. Frequency sweeps were performed over a range between 0.1 and 100 rad/s. Temperature was maintained at 25 °C via the use of Peltier plate control. In order to minimise disruption to the physical


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Biomacromolecules

hydrogels, a cone-plate geometry (40 mm, 2 º 1’ 37”) with a gap of 51 µm was used. Samples were loaded and trimmed with a spatula. A water trap was used to minimise evaporation. Measurements were performed in triplicate to minimise the effects of loading artefacts. Atomic force microscopy Atomic force microscopy images of the samples were obtained using a Multimode 8 (Bruker BioSciences Corporation). For sample preparation, peptide hydrogels (10 mg/mL) were diluted to a final peptide concentration of 0.5 mg/mL, before 15 µL of each diluted solution was applied to highly ordered pyrolytic graphite (HOPG) substrates (SPI), with any redundant samples being absorbed by pipette. The tips used were ScanAsyst-Air probes with silicon tip on nitride lever (Bruker BioSciences Corporation). The scan was operated in PeakForce QNM imaging mode. Scan size was at 10 µm. Transmission electron microscopy JEOL-2100 LaB6 transmission electron microscopy (JEOL Ltd.) at an operation voltage of 100 kV was used for imaging. Agar lacey carbon coated films on 300 mesh copper grids (Emgrid Pty. Ltd.) were used as sample holders. For sample preparation, peptide hydrogels were diluted to 0.1 mg/mL. The sample (12 µL) was applied onto the grid and allowed to absorb for 5 min before excess fluid was wicked off using split Whatman filter paper (No.1). One drop of negative stain NanoVan (Bio-Scientific Pty. Ltd.) was put onto parafilm “M”, then the grid was placed on the stain with carbon side down, allowed to stain for 2 min, then air dried for 2 min with the carbon side up. Finally, the grids were placed into a grid box to dry overnight. HEK 293T and C2C12 myoblast cell culture and conditioned media preparation


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HEK 293T cells were cultured in DMEM containing 10% (v/v) FBS and 0.4% (v/v) pen/strep in atmospheric O2 plus 5% CO2 at 37 °C. Cells were transfected using Lipofectamine 2000 (Life Technologies) with constructs encoding the V1 versican construct (provided by Professor Dieter Zimmermann), and empty vector control (pcDNA3.1MycHisA, Life Technologies). Serum free conditioned medium was produced as previously described56. The conditioned media was collected into 1.5 mL Eppendorf tubes at 48 h post transfection and stored at -80 °C. Just prior to use, 10% FBS was added to the conditioned media. C2C12 myoblasts were cultured in growth media (25 mM glucose DMEM plus 10% FBS) in atmospheric O2 with 5% CO2 at 37 °C and passaged when 70%-80% confluent. For all experimental analysis, the 400 µL of hydrogels were assembled or co-assembled in flat bottom 24-well tissue culture plates before 200 µL C2C12 myoblasts, suspended in growth media, were added to gels at a cell density of 20,000 cell/cm2. Western blot of versican enriched conditioned media Chondroitinase ABC was added to an aliquot of conditioned media to deglycosylate versican and improve the binding affinity of the V1/V0 versican anti-GAG-β antibody (AB1033, Merck Millipore) to V1 versican. To prepare the conditioned media samples for immunoblotting, 1 µL chondroitinase ABC was added to 29 µL of versican or empty vector conditioned media and incubated at 37 °C for 2 h 57. The samples were then diluted with a 4× loading buffer (BioRad) containing β-mercaptoethanol and incubated at 95 °C for 5 min. Samples were run on miniprotean TGX stain-free gels (BioRad) at 120 V. Gels were then activated and proteins visualised using the ChemiDoc XRS+ system (BioRad). The gels were transferred to PVDF membranes using the Trans-Blot Turbo system (BioRad), as per manufacturer’s recommendations. PVDF


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membranes were blocked in 5% (w/v) skim milk in TBST for 1 h at room temperature, then incubated with versican anti-GAG-β antibody (AB1033, Merck Millipore) (1:500 dilution in 1% skim milk) overnight at 4 °C, followed by a 1 h incubation with a secondary goat anti-rabbit HRP antibody in 1% skim milk in TBST (1:5000). The bands were detected using ECL substrate (BioRad) and blots were imaged on the ChemiDoc XRS+ system. Immunofluorescence to confirm the incorporation of versican into the hydrogel scaffolds Hydrogels were co-assembled with versican or empty vector conditioned media. The aliquots of gel were dried onto poly-L-lysine microscope slides and allowed to air dry. Scaffolds were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 5 min at room temperature before proceeding with immunostaining using the V1/V0 versican anti-GAG-β antibody, as previously described

48

. Stained gels were imaged on a bench-top confocal microscope

(Fluoview FV10i, Olympus) at 600x magnification. Live/dead cell staining C2C12 cells were subjected to live/dead cell staining using calcein AM and propidium iodide at 24 and 72 h post seeding onto the four groups of hydrogels (Sapphire Bioscience Pty. Ltd.), as per manufacturer’s instructions. Following a 60 min incubation in atmospheric O2 with 5% CO2 at 37 °C, live and dead cells were imaged using a ZOETM fluorescence cell imager (BioRad) at 200x magnification. Live cells stained green with calcein AM and dead cells stained red with propidium iodide. The cell size, as an indicator of cell spreading, was measured using ImageJ. N = 3 biological replicates performed in duplicate wells for each scaffold at both time points, and in total cell area was determined for an average of 880 cells per scaffold.


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Myoblast number and growth Cell number was assessed at 72 h post seeding using a commercially available kit (Vybrant® Cytotoxicity Assay Kit (Glucose-6-phosphate dehydrogenase (G6PD) Release Assay, V231111, ThermoFisher Scientific), as per manufacturer’s instructions with the following modifications. The hydrogels were solubilised in 15 mL PBS and centrifuged at 3202 g for 5 min at room temperature to pellet the cells. Then 12 mL of PBS was removed, and the myoblasts were washed and centrifuged a second time in 15 mL PBS. The resultant cell pellet was resuspended 1 mL of PBS, and centrifuged at 11688 g for 5 min at room temperature. The cell pellet was lysed in 1% lysis buffer for 15 min on ice and diluted 1:10 to assay glucose-6-phosphate activity, a marker of cell number, using a Multi-Mode Micro Plate Reader Flexstation II

384

(Molecular

Devices) at a 544 nm excitation wavelength and a 590 nm emission wavelength. The cell lysate prepared for the Vybrant Cytotoxicity Assay Kit, was also used to assess total protein content, as an additional marker of cell growth. This was done using a Pierce BCA Protein Assay Kit (ThermoFisher Scientific), as per manufacturer’s instructions. N = 3 biological replicates were performed in triplicate wells for each experiment and the results were normalised to the respective control scaffolds, Fmoc-FRGDF or Fmoc-FRGDF co-assembled with empty vector conditioned media. Nuclei and actin staining to assess cell structure and the formation of multinucleated syncytia C2C12 myoblasts were stained with Rhodamine phalloidin (Life Technologies) to visualise actin and the nuclear stain DAPI (Life Technologies) at 72 h post seeding to assess the effects of the hydrogels on cell morphology and the formation of multinucleated syncytia. Briefly, myoblasts


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and hydrogels were fixed in 200 µL of 4% paraformaldehyde in PBS (ProSciTech Pty. Ltd.) for 20 min at room temperature, then washed in PBS and solubilised in 200 µL of 0.1% Triton × 100 (ProSciTech Pty. Ltd.) for 15 min. The cells were then stained with DAPI (1:200 dilution in PBS) and phalloidin (1:150 dilution in PBS) for 60 min. Cells were imaged using ZOETM fluorescence cell imager (BioRad) at 200x magnification. The number of myonuclei in a cell or syncytium was counted using ImageJ. N = 3 biological replicates were performed in duplicate wells for each scaffold at 24 and 72 h post seeding. In total the number of nuclei was determined for an average of 450 cells or syncytia per scaffold. To support the cell size data obtained with calcein AM stain, in 2 of the 3 biological replicates, cell size was determined using ImageProPlus. Briefly, a reverse mask was used to identify fluorescently labelled red and blue cells. This was done by selecting the black background. Then the software automatically counted the cells of interest and the area of the cells was determined. Cells at the edge of the image were excluded and a minimum area of 40 µm2 was set. Statistical Analysis For the cell culture experiments, results are expressed as mean ± S.E.M. and significance was calculated using a Student paired, two tailed t test between control and test groups. Data were considered statistically significant when a p value < 0.05 was obtained. Results and Discussion The formation of hydrogels Fibronectin binds to numerous cell surface receptors and ECM proteins, including integrins, collagens and proteoglycans. This can give rise to a variety of cellular responses

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. The


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tripeptide sequence RGD, derived from fibronectin, can promote cell attachment

59

. For most

materials functionalised with RGD, the RGD domain will not act in isolation, a critical issue which has not been sufficiently addressed

59

. Combining RGD with proteoglycan-binding

peptides has in some instances facilitated cell and material association

60

. Therefore, in this

study, the minimalist pentapeptide Fmoc-FRGDF (Figure 1A), containing a biologically available sequence RGD, was synthesised using the SPPS method to yield a crystal white peptide powder, as previously reported

25, 61

. Four groups of hydrogels were prepared using this Fmoc-

SAP: (1) Fmoc-FRGDF, (2) + Fucoidan, (3) + empty vector conditioned media and (4) + versican conditioned media. Among them, hydrogel (1) is a self-assembly system, and hydrogels (2-4) are co-assembly systems. The self-assembly and co-assembly processes of these SAPs were triggered using a well-characterised pH switch method at a peptide concentration of 1.0 wt%

61

(Figure 1B, D & F). In the co-assembled system 2, the readily dissolved fucoidan

(Figure 1C) powder (0.5 mg/mL) was mixed with the peptide powder, then co-assembly was initiated by pH change (Figure 1D). The Fmoc-FRGDF hydrogels were used as a control of fucoidan hydrogels. Serum free versican or empty vector conditioned media was obtained from HEK 293T cells transfected with a V1 versican construct or an empty vector (pcDNA3.1MycHisA) respectively 48 (Figure 1F).

Confirmation of V1 construct transfection effectiveness The incorporation of fucoidan with peptide nanofibrils has been confirmed in our previous work 25

. To assess the incorporation of versican into the Fmoc-FRGDF scaffold, the effectiveness of

V1 versican production by transfected HEK 293T cells was confirmed using western blotting


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(Figure 2A). As expected, a prominent, high molecular weight band, greater than 250 kDa , was observed in the versican conditioned media, and was absent in the empty vector conditioned media

62

. A second 70 kDa band was also detected in versican conditioned media. This band

corresponds to versikine versicanases

62

63

, which is produced by proteolysis of versican by ECM ADAMTS

. The presence of protein bands in the versican and empty vector lanes of the

SDS-PAGE gel indicates that the HEK 293T cells secrete proteins other than versican. Hence, it was necessary to use empty vector conditioned media as a control (Figure 2B). To confirm the inclusion of versican by co-assembly with conditioned media and the SAP hydrogel, immunofluorescent staining with the anti-GAG-β versican antibody, and confocal imaging, were both used to visualise versican within the hydrogel (Figure 2B). Versican was not detected in the empty vector conditioned media-SAP system (Figure 2B-a). However, in the scaffold co-assembled with versican conditioned media, the staining with the anti-GAG-β versican antibody indicated the presence of versican and its localisation to the peptide nanofibres (red) (Figure 2B-b), which were distributed in a 3D arrangement (Figure 2B-c) and indicates the successful inclusion of versican into the hydrogel.

Investigation of nano- and microstructures of hydrogels To characterise how the addition of fucoidan, versican or empty vector conditioned media affected the nano- and microstructure of the resultant hydrogels, TEM and AFM imaging were used. It should be noted that these are dried samples. Care has been taken to use representative images, as artefacts can be generated during the drying process64. TEM images (Figure 3A-D) show that in the four groups of hydrogels, nanofibrils ~10 nm in diameter and microns in length


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were formed, which is in line with previous measurements using SANS51. AFM images indicate that in the Fmoc-FRGDF hydrogel, the nanofibrils interweaved and formed a homogenous network, as previously observed 61. With a fucoidan concentration of 0.5 mg/mL, some ’bundle’ formation was observed (Figure 3E & F). . We have previously observed that the presence of macromolecules can affect the ordering of the individual fibrils into bundles. At higher fucoidan concentrations of 5 and 10 mg/mL, individual fibrils came together to form large, highly aligned ‘bundles’. Here, this effect was conserved at low fucoidan concentrations51. In the hydrogels coassembled with the versican conditioned media or the empty vector conditioned media, the co-assembly process formed scaffold morphologies which are different compared to the Fmoc-FRGDF and fucoidan scaffolds. The fibrils appeared less linear and more flexible, with increased densification (Figure 3G&H). It is likely that proteins found in FBS and macromolecules secreted by HEK 293T cells contribute to the different morphology of these scaffolds and are all included into the scaffold during formation. This has been previously observed when composites are formed with the basement membrane protein, laminin50 and with the complex polysaccharide dextran65. Characterisation of the co-assembled structure, mechanism and mechanical properties To assess whether the addition of fucoidan, versican and empty vector conditioned media affected the self-assembly mechanism of the Fmoc-FRGDF scaffold, the secondary structures were characterised using FTIR and CD, and the bulk mechanical properties of the scaffolds were determined via oscillatory rheometry. As shown in Figure 4A, the FTIR spectrum has a large single peak at 1630 cm-1 in the amide I region in all the four systems, indicating the presence of a consistent peptide arrangement, based around β-sheet interactions The small shoulder peak at 1690 cm-1 is attributed to a stacked carbamate group.66-67. Zanuy and coworkers have performed


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Biomacromolecules

atomistic molecular dynamics simulations of RGD containing Fmoc-peptides, and have shown these longer peptides can form hierarchical assemblies, involving a rich variety of intermolecular interactions, forming a highly packed network of inter-backbone hydrogen bonds, which in turn stabilise the structure68 Importantly, there are no shifts of the peak at 1630 cm-1 in the coassembly systems, which shows that the addition of the macromolecules fucoidan, versican or the components of the empty vector conditioned media does not interrupt the broad selfassembly mechanism. For CD measurements, a whole gel suspension was prepared in order to reduce the scattering from the hydrogel. The CD spectrum (Figure 4B), the large transition at ~220 nm shows the Cotton effect induced by n–π* transition, and provides further confirmation of β-sheet based structure expected from the assembly 69, reinforcing the FTIR results. The broad transition centred at 260 nm shows an increase in magnitude, driven by increased supramolecular ordering of nanofibrils, agreeing with other similar assemblies 15, 70. The viscoelastic properties of the hydrogels were tested using cone-plate rheological analysis (Figure 4C & D). Results showed that in each system, the elastic modulus, G’, is greater than the viscous modulus, G”, over a range of frequencies, indicating a self-supporting hydrogel. Moreover, the co-assembly with fucoidan (0.5 mg/mL), versican and empty vector conditioned media increased the stiffness of the hydrogels by approximately one order of magnitude. For the fucoidan system, we anticipate that the increase in elastic moduli directly results from interactions between the fucoidan and the peptide fibrils forming dense bundles. The stiffness of versican and empty vector systems were comparable. The proteins and biomolecules secreted from HEK 293T cells from both versican and empty vector conditioned media likely contributed to the formation of stiffer hydrogels. We have demonstrated previously that the incorporation of biomolecules led to increased fiber alignment, bundling and densification of the fibrils25, 51. As


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the fibril structure is maintained, we believe this process results in the larger bundles becoming stiffer and the junction points between these structures become reinforced by the additional interactions between the biomacromolecules and the fibrillar structures. This ‘molecular glue’ effect results in the scaffolds becoming stiffer through increased junction points in the structure on a microscale level. Importantly, there was no significant difference between stiffness in the fucoidan, empty vector and versican hydrogels. This suggests that the mechanism was conserved across all three conditions. Any differences in biological activity would therefore not be an effect of the stiffness of the scaffold.

Effect of co-assembled Fmoc-SAP scaffolds on cell number and growth The biological compatibility and activity of the co-assembled scaffolds were investigated in vitro using C2C12 myoblasts. Following 72 h in culture, cell number was similar between myoblasts seeded on the Fmoc-FRGDF scaffolds and fucoidan containing scaffolds (Figure 5A). This observation is in accordance with findings of Lee, et al., where soluble fucoidan had no effect on C2C12 cell viability during myogenic differentiation at concentrations up to 0.1 mg/mL; an effect that seems to be conserved in the immobilised molecule 54. The total protein content was also similar between myoblasts cultured on the Fmoc-FRGDF scaffolds and fucoidan containing scaffolds (Figure 5B), suggesting that cell number and/or protein synthesis were not affected by immobilised fucoidan. The chondroitin sulphate proteoglycan versican was selected as a candidate ECM molecule, as it is highly expressed during development in skeletal muscle as part of a provisional matrix

44, 46

and is thought to stimulate the proliferation and differentiation of muscle progenitor cells

46

.


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Biomacromolecules

Versican is remodelled by ADAMTS versicanases to produce a bioactive 70 kD peptide fragment known as versikine 63. This remodelling of versican is required for myoblast fusion and myofibre formation apoptosis

48

45

. In another biological content, versikine has been shown to stimulate

, which may have implications for viability if cells are cultured in a hydrogel co-

assembled with versican. Since C2C12 myoblasts synthesise and secrete ADAMTS versicanases 45

, it is quite likely that the versican incorporated into the scaffolds may be remodelled into

versikine. Versikine fragments were also detected in the conditioned media and presumably incorporated into the scaffolds (Figure 2B). Here, versican had no effect on cell number or growth as cell lysate G6PD activity, a marker of cell number, was similar between C2C12 myoblasts cultured on versican and the empty vector scaffolds (Figure 5C), as was total protein content (Figure 5D).

Effect of co-assembled Fmoc-SAP scaffolds on cell size and spreading Cell spreading has been shown to be a marker of attachment to the hydrogel71, therefore Calcein AM/propidium iodine live/dead staining was used to assess cell morphology and integration with the scaffolds at 24 h and 72 h post seeding, and as a qualitative marker of cell viability. Cells migrated into all four hydrogels and distributed evenly throughout the volume of the hydrogel. In all scaffolds, C2C12 myoblasts displayed high levels of viability as indicated by the very low level of red PI staining (Figure 6A). This demonstrates the utility of this approach, as previous studies on the unfunctionalised and sub-optimal Fmoc-FF hydrogels report gel collapse leading cell death within 48 hours22. Viable myoblasts, which are stained green by calcein AM, displayed a rounded morphology on all four scaffolds at 24 h post seeding. However, by 72 h in


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the Fmoc-FRGDF, versican, and empty vector scaffolds, the cells displayed numerous prominent cytoplasmic projections and increased cell spreading. Conversely, myoblasts in the fucoidan hydrogel were small, with a rounded or spindle shaped morphology with fewer cytoplasmic projections. To more thoroughly assess the effects of fucoidan and versican incorporation on myoblast size and spreading, computerised image analysis was performed. Between 24 h to 72 h post seeding, the average cell size increased by 225% (p < 0.001), 465% (p < 0.001) and 1227% (p < 0.001), in Fmoc-FRGDF, empty vector and versican scaffolds respectively, whilst the average cell size in the fucoidan hydrogel did not increase.

At 24 h post seeding, there were more ‘small’ cells with an area 10 nuclei (p < 0.001) was lower in the fucoidan scaffold compared to the Fmoc-FRGDF control scaffold. Furthermore, the fucoidan scaffold had significantly more mononuclear C2C12 myoblasts (p < 0.001) (Figure 7A&B). It is possible that incorporation of fucoidan into the scaffolds limited myogenic differentiation into multinucleated cells due to limited spreading. Indeed, Lee, et al. have shown that the addition of fucoidan to C2C12 cell culture media reduced the expression of the myogenic regulatory factors and inhibited the morphological changes that occur during differentiation 54. Unlike fucoidan, by 72 h post seeding the formation of multinucleated syncytia was similar in scaffolds containing versican or empty vector conditioned media (Figure 7A & &C). In cultured C2C12 myoblasts, excess versican inhibited myoblast fusion into multinucleated muscles fibres. 45

.

45

The fact that versican incorporation into scaffolds did not decrease the number of

multinucleated muscle cells might be attributed to versican being degraded by ADAMTS versicanases, as discussed above

45

. From DAPI staining, it is also evident that total myonuclei

number per image was unchanged, confirming our conclusion that the biomacromolecules, once incorporated to the scaffolds do not affect cell viability or proliferation, only cellular processes. Conclusions The co-assembled SAP scaffolds with fucoidan or versican conditioned media showed a conserved mechanism of assembly and final scaffold, yet demonstrated distinct structural morphologies and biological effects on C2C12 myoblasts cultured within them. Importantly, the incorporation of fucoidan, versican conditioned media, or empty vector conditioned media into


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the Fmoc-FRGDF scaffold did not interrupt the self-assembly mechanism. Co-assembly using fucoidan, versican, and empty vector conditioned media however resulted in roughly a 10-fold increase in stiffness compared to the Fmoc-FRGDF hydrogel, suggesting that these are effective mechanisms to tune the mechanical properties of these scaffolds. Interestingly, when culturing C2C12 myoblasts in the four scaffolds, cell viabilities were similar for up to 72 h post seeding. However, myoblasts grown in the fucoidan scaffold were smaller in size at both 24 h and 72 h post seeding and the formation of multinucleated synthetia was reduced, whilst the total protein content did not differ between scaffolds. Altogether, these observations indicate that the incorporation of fucoidan into the scaffold limits the spreading of C2C12 myoblasts. For myoblasts cultured on the versican scaffold, a higher number of smaller cells, with no difference in protein content, were observed at 24 h, but not at 72 h post seeding when compared to myoblasts seeded on the empty vector scaffold. We hypothesise that this might be attributed to the remodelling of versican by extracellular matrix versicanases secreted by the C2C12 myoblasts. In conclusion, these co-assembled scaffolds are biocompatible, and through the incorporation of different biomacromolecules, have distinct effects on cell behaviour. Our observations highlight the potential of these scaffolds as tunable, biomaterial candidates for future 3D stem cell culture work.

FIGURES


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Biomacromolecules

Figure 1. Cartoon of the self-assembly and co-assembly mechanisms, and the final hydrogels. (A) The chemical structure of Fmoc-FRGDF. (B) Diagram for the self-assembly of FmocFRGDF. Demonstrating the stacking of Fmoc groups through π-π interactions, and the interlocking of peptide sequences through β-sheet formation - subsequently forming nanofibrils, which interweave to form a scaffold. (C) The chemical structure of the low molecular weight fucoidan subunit. (D) Schematic of co-assembly of Fmoc-FRGDF with fucoidan. (E) A schematic of V1 versican, note the G1 and G3 globular domains and the chondroitin sulphate side chains bound to the GAG-β subunit. (F) Co-assembly of Fmoc-FRGDF with versican conditioned media or empty vector conditioned media, resulting in these molecules being present on the surface of the peptide nanofibrils. (G) Self-supporting hydrogel of (i) Fmoc-FRGDF; (ii) + Fucoidan, (iii) + Empty vector conditioned media and (iv) + Versican conditioned media.


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Figure 2. Confirmation of versican production by HEK 293T cells and versican incorporation in Fmoc-FRGDF hydrogel. (A) Western blot demonstrating the presence of versican in the versican transfected, but not empty vector transfected, conditioned media. A prominent band, greater than 250 KDa, shows the core protein residue of chondroitinase ABC cleaved versican. The smaller ~ 70 kDa fragment is likely cleaved versikine. (B) Confocal image showing labeling of V1 versican complex in the hydrogel with the V1/V0 versican anti-GAG-β antibody (red). (B-a) Untransfected conditioned media-SAP hydrogel; a ̀ corresponding bright field image. (B-b) Transfected V1 versican conditioned media-SAP hydrogel, b ̀ corresponding bright field image. (B-c) Z-stack of b showing three-dimensional fibres.


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Biomacromolecules

Figure 3. Nano and micro morphologies of hydrogels. TEM images indicate nanofibers formed by (A) Fmoc-FRGDF, (B) + Fucoidan, (C) + empty vector and (D) + versican hydrogels. (E-H) Equivalent samples of AFM images show the micro structure.


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Figure 4. Secondary molecular packing and mechanical properties. (A) FIIR spectrogram in the amide I region shows the presence of antiparallel β-sheet structure. (B) CD spectrogram confirms the antiparallel β-sheet structure and also shows the supramolecular ordering of nanofibrils in each hydrogel. (C) Elastic and Viscous moduli over a range of frequencies of hydrogels formed by Fmoc-FRGDF and + Fucoidan and (D) Fmoc-FRGDF + versican conditioned media and + empty vector conditioned media


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Biomacromolecules

Figure 5. Cell number and growth 72 h post seeding. (A) Relative cell number and (B) relative total protein content, a marker for growth, of cells seeded in Fmoc-FRGDF and + Fucoidan hydrogels. Data was normalised to the Fmoc-FRGDF group. (C) Relative cell viability and (D) relative total protein content of cells seeded in + Empty vector and + Versican hydrogels. Data were normalised to the empty vector group.


31

Biomacromolecules

A

24 h

200µm

200µm

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72 h

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C 45%

***

40%

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*

25%

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15%

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Cell spreading area (µm2) 45% + Empty vector-Day 1

40%

+ Versican-Day 1

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Cell spreading area (µm2)

E 45%

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Large and Small assembly: Combining functional macromolecules

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