Encoding Stem Cell-Secreted Extracellular Matrix Protein Capture in

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Encoding Stem Cell-Secreted Extracellular Matrix Protein Capture in Two and Three Dimensions Using Protein Binding Peptides Hadi Hezaveh, Steffen Cosson, Ellen A. Otte, Guannan Su, Benjamin D. Fairbanks, and Justin J. Cooper-White Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01482 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Encoding Stem Cell-Secreted Extracellular Matrix Protein Capture in Two and Three Dimensions Using Protein Binding Peptides Hadi Hezaveha,b, Steffen Cossona,b, Ellen A. Ottea,b, Guannan Sua,b, Benjamin D. Fairbanksb and Justin J. Cooper-White a,b,c, * a

Tissue Engineering and Microfluidics Laboratory, The Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, St. Lucia QLD, Australia

b

Commonwealth Scientific and Industrial Research Organization (CSIRO), Manufacturing Flagship, Clayton, Victoria 3169, Australia c School of Chemical Engineering, University of Queensland, St. Lucia QLD, Australia *

Corresponding author: [email protected]

Abstract Capturing cell-secreted extracellular matrix (ECM) proteins through cooperative binding with high specificity and affinity is an important function of native tissue matrices during both tissue homeostasis and repair. However, whilst synthetic hydrogels, such as those based on polyethylene glycol (PEG), are often proposed as ideal materials to deliver human mesenchymal stem cells (hMSCs) to sites of injury to enable tissue repair, they do not have this capability, a capability that would enable cells to actively remodel their local extracellular microenvironment and potentially provide the required feedback control for more effective tissue genesis. In this work, we detail a methodology that engenders polyethylene glycol (PEG)-based two-dimensional substrates and three-dimensional porous hydrogels with the ability to capture desired extracellular matrix (ECM) proteins with high specificity. This ‘encoded’ ECM protein capture is achieved by decorating the PEG-based


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materials with protein binding peptides (PBPs) synthesised to be specific in their binding of fibronectin, laminin and collagen I, which are not only the most omnipresent ECM proteins in human tissues, but as we confirmed, are also secreted to differing extents by hMSCs under in vitro maintenance conditions. By encapsulating hMSCs into these PBPfunctionalised hydrogels, and culturing them in protein-free maintenance media, we demonstrate that these PBPs not only actively recruit targeted ECM proteins as they are secreted from hMSCs, but retain them to much higher levels compared to nonfunctionalised gels. This novel approach thus enables the fabrication of encoded surfaces and hydrogels that capture cell-secreted proteins, with high specificity and affinity, in a programmable manner, ready for applications in many bioengineering applications, including bioactive surface coatings, bioassays, stem cell culture, tissue engineering and regenerative medicine.

Key Words Poly (ethylene glycol) hydrogels, mesenchymal stem cells, protein binding peptide, extracellular matrix, remodelling.

1. Introduction Extracellular matrix (ECM) proteins are an essential component of any stem cell niche within tissues, as they directly (through integrin-mediated interactions), and indirectly (through cooperative binding interactions with other ECM components) modulate the maintenance,



proliferation, self-renewal and differentiation of stem cells1. This dependence on ECM composition is paralleled in vitro, where it is clear that mesenchymal stem cells (MSCs) produce a range of different ECM proteins as they differentiate (in response to inductive media) into defined tissue cell types2. Furthermore, it has also been shown that the type and amount of ECM presented to them during in vitro culture can actively drive these stem cells to bias fate choice, including towards specific differentiated cell types3. In an effort to take advantage of this ‘feed-forward’ control loop, biomaterial scientists have for some time incorporated full length ECM proteins, or fragments/peptides thereof, into synthetic surfaces or hydrogels to create both 2D and 3D bio-synthetic microenvironments representative (in terms of ECM composition and architecture) of different tissues or stem cell niches4, 5. In this endeavour, poly(ethylene glycol) (PEG)-based biomaterials are one of the more commonly utilised systems, due to their favourable property slate, including their bio-inert and non-fouling properties6, as well as the ease with which end-group functionalisation can be achieved. Functionalisation of PEG chain ends can be utilised to introduce crosslinking moieties to enable facile formation of hydrogels from linear or branched PEG macromers. A plethora of crosslinking schemes have been developed7 in order to generate hydrogels that mimic biochemical and/or biophysical matrix properties of the ECM. Of these, photo-initiated crosslinking offers temporal and spatial control over gelation, allowing mechanical and/or chemical patterning of PEG hydrogels, further enhancing their ability to mimic matrix biology8-10. Moreover, photo-initiated click chemistry utilising norbornene11 offers additional advantages, including crosslinking under mild conditions and the highest reaction rate of any click chemistry system investigated to date10. These same pendant end-groups have


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been used to incorporate bioactive moieties, including proteins and peptides derived therefrom, so as to introduce biochemical cues matching those seen by stem cells in their native tissue microenvironments.12 Coupling of these bioactive elements has been achieved using several reaction schemes, including click chemistry13 and Michael’s addition of cysteine residues (with in the proteins or peptides) to vinyl sulfones or maleimides in the pH range of 6.5-7.514, 15. Protein-derived peptides have been shown to engender a biomaterial surface or hydrogel with various biomimetic functions, including enabling cell adhesion and growth factor binding 16. However, a static ECM protein composition of the cellular microenvironment does not reflect the highly dynamic conditions within tissues, even if we consider homeostasis, where the ECM is continually being degraded and secreted (and thus soon incorporated into surrounding networks) by the cells. Importantly, this ‘remodelling’ is a necessary response post tissue injury and during repair. In vivo, the ECM permits this by having co-operative, highly specific interactions between different ECM proteins. These encoded interactions permit the dynamic formation of new three-dimensional networks; even as existing networks are being degraded. Whilst currently available PEG-based hydrogels have been adapted to mimic many of the characteristics of native ECM, they do not have this capability, and moreover, they are either quite effective at avoiding protein binding and hence distributing (through diffusion) these secreted ECMs far from the cells, or achieve it through non-specific interactions17. Ideally, by design, bio-synthetic surfaces and hydrogels targeted for stem cell culture and tissue regeneration applications, respectively, should include elements that enable cells to actively modulate the composition of their ECM microenvironment through their own secretions over time.



In this paper, the concept of encoded ECM protein recruitment on biomaterial surfaces and throughout 3D hydrogels using protein binding peptides is developed. To achieve this, we confirmed the presence, and characterise the relative amounts of the most abundant ECM proteins found within tissues - fibronectin, collagen I and laminin - deposited during MSC culture under maintenance conditions. Next, protein binding peptide (PBP) sequences found within extracellular matrix proteins and shown to bind to fibronectin, collagen I and laminin18-20 were synthesised. The binding affinity and specificity of these peptides to their respective ECM partners were thoroughly characterised. Norbornene-functionalised PEGbased hydrogels were conjugated with the validated protein binding peptides (PBPs) and their architecture, mechanics, and ability of the presented PBPs to capture ECM from the surrounding fluid environment was assessed. Lastly, we performed extended culture of hMSCs encapsulated in each of these PBP-functionalised hydrogels under maintenance conditions. Our results confirm that the proposed combination of a proven cell-supportive PEG-based hydrogel with protein binding peptides provides a novel platform with which to capture secreted proteins with specificity at surfaces and throughout 3D hydrogels, opening up the possibility of harnessing the endogenous process of cell-driven tissue remodelling during homeostasis and repair and, ultimately, to selectively drive differentiation post implantation.

2. Materials and Methods 2.1 Materials Rink-amide resin, piperidine, anhydrous N, Nˊ dimethylformamide (DMF), O- (Benzotriazol1-yl)-N,N,Nˊ,Nˊ tetramethyluroniumhexafluorophosphate (HBTU), triisopropylsilane (TIPS), Dichloromethane (DCM), N, Nˊ dimethylformamide (DMF), diethylether diethyl ether and


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trifluoroacetic acid (TFA), NHS-PEG-Maleimade (Mw=5000 Da), human collagen I and human laminin were purchased from Sigma-Aldrich. Dithiothreitol (DTT) was purchased from Research Products International. Human fibronectin was purchased from Corning. All amino acids were provided from Mimotopes. QCM-D chips were purchased from Q-Sense, Sweden. Cysteamine was provided by Alfa Aessar. 8-arm PEG-hydroxyl (10kDa) (Jenkem), 4-arm PEG thiol (10kDa) (JenKem), Norbornene carboxylic acid (Sigma Aldrich), Dichloromethane (DCM) and N,N’-Dicyclohexylcarbodiimide (DCC) (Sigma Aldrich), Human mesenchymal stem cells (lonza), Low glucose DMEM and FBP (Gibco, 10567-022), Hoescht 33342, Phalloidin (Alexa Fluor 488) and secondary antibody, Alexa Fluor 647 goat anti mouse IgG1 (γ1) (Invitrogen), were all used as received from suppliers.

2.2 Methods 2.2.1 Maintenance and Expansion of hMSCs Human bone marrow-derived mesenchymal stem cells (hMSCs) from the supplier (Lonza) were seeded at a density of 5000 cell/well and sub-cultured in low glucose DMEM (Gibco, 10567-022) with 10% FBS at 37˚C in a humidified atmosphere of 5% CO2, until 80% confluence, at which time they were passaged. Cells used in all experiments detailed in this work were between passage 3 and 6.

2.2.2 hMSC extracellular matrix deposition on tissue culture plate (TCP) surfaces Culture hMSCs were seeded at 15,000 cells/cm2 and cultured under maintenance conditions on 96 well plates in either a.) standard maintenance media (low glucose DMEM with 10% FBS) for up to 21 days or b.) animal component/serum-free media (Mesencult-ACF culture Kit) for up to 7 days, to investigate ECM secretion.



2.2.3 Immunofluorescence Staining Immunofluorescence staining was performed on fixed samples (4% Paraformaldehyde) using a mouse monoclonal α-fibronectin antibody (Sigma Aldrich, F0916), a mouse monoclonal α-Collagen type 1 antibody (Sigma Aldrich, C2456), a mouse monoclonal αLaminin antibody (Sigma Aldrich, L8271) and Anti-mouse IgG1 secondary antibody conjugated with Alexa Fluor 647 (Life Technologies, A21121). 1% BSA was used to block samples overnight prior to staining. 2.2.4 Imaging and Analysis Images of cultured hMSCs and their ECM protein secretions on TCP were taken with a high content fluorescence microscope (Operetta, PerkinElmer) using a 20X 1.7 WD objective. Quantification of the relative amounts of each ECM protein was by fluorescence intensity, quantified using Harmony 4.1 software (PerkinElmer).

2.2.5 Peptide synthesis, purification and characterisation 2.2.5.1

Synthesis

All peptides were prepared by solid phase peptide synthesis (SPPS) on solid Rink-amide resin using Fmoc chemistry at the 0.2 mmol scale. 20% piperidine in N, Nˊ dimethylformamide (DMF) was used for deprotection of Fmoc group. Subsequent Fmoc protected amino acids were coupled to the free amino group using O- (Benzotriazol-1-yl)-N,N,Nˊ,Nˊ tetramethyluroniumhexafluorophosphate (HBTU) as the coupling reagent. After each deprotection and coupling step, the resin was washed with DMF (~5mL) 6 times for 3 minutes. After coupling the last amino acid, excess reagents were removed by washing 6 times in DMF for 5 minutes followed by five steps of washing using DCM for 2 min (~5 mL). The resin was then vacuum dried for 2 hours prior to cleavage. Peptides were cleaved from


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the resin and deprotected in trifluoroacetic acid (TFA) with 2.5% deionized water, 1.25% dithiothreitol (DTT, Research Products International) and 1% triisopropylsilane (TIPS) under nitrogen flow for 2hrs. Peptides were then precipitated in 20 mL per gram of resin of icecold diethyl ether and centrifuged at 4˚C at 4400 rpm for 5 minutes. The precipitate was washed with diethyl ether before resuspension in deionized water. The solution was frozen with liquid nitrogen and lyophilized overnight to yield a fluffy, white powder. 2.2.5.2

High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) (HPLC; Agilent Technology 1260 Infinity) in an acetonitrile/water gradient under acidic conditions on a Phenomenex Luna 5um C18 (2) column (5 µm pore size, 100 Å particle size, 150×4.6 mm) was used to check the presence of the main peak representing peptide and other components (impurities). Preparative HPLC (Agilent Technology 1260 infinity) with Phenomenex Luna 10um C18 (2) column (10 µm pore size, 100 Å particle size, 150×21.2 mm) column was used to separate peaks and purify the peptide. 2.2.5.3

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS)

MALDI-TOF MS measurements were performed on a Bruker Autoflex III MALDI-TOF mass spectrometer. The purified samples were dissolved in water at a concentration of 1 mg.mL-1. A stock solution of the matrix was prepared by dissolving 10 mg HCCA-ALPHA-Cyano-4hydroxycinnamic acid in 1 mL water. The sample and matrix solutions were combined in the ratio of 100 μL to 10 μL, respectively. A 1 μL aliquot of the mixture was then applied to the PAC (Pre-spotted Anchor Chip) MALDI plate and air-dried at ambient temperature (20 ˚C). Measurements were performed at an acceleration voltage of 19 kV, in positive ion reflection mode. Suitable values for laser power, gain and laser shots were determined for each



sample to produce the best quality data (best resolution, reduced fragmentation and ion statistics). 2.2.6 Quartz crystal microbalance with dissipation (QCM-D) Peptide binding affinity to ECM proteins was measured using a quartz crystal microbalance with dissipation monitoring (QCM-D E4, Q-Sense AB). The diameter of chips was 14mm and the thickness 0.3mm (Q-Sense E4, Gothenburg, Sweden). Clean gold-coated QCM crystals were immersed in a 10mM cysteamine solution for 2 hours at room temperature under nitrogen flow. After rinsing with DI water and drying with N2, the crystals were mounted inside QCM-D flow modules. For each measurement, phosphate buffered saline (PBS) buffer at pH 7.4 was first added to the chambers at a rate of 20 µL/min (at 20 ◦C) to obtain stable baselines. Then, the first layer (NHS-PEG-Maleimide) with a concentration of 0.3 µM in PBS was introduced, followed by washing with PBS. The peptide solution with a concentration of 1 mM was subsequently reacted within the chamber and then washed. Finally, protein (0.1 µM) was flowed over, again followed by a washing step. Shifts in the crystal frequency and dissipation were recorded using Q-Soft software (Q-Sense AB). The change in oscillator frequency was used to estimate the amount of adsorbed material, based on the Sauerbrey equation, as follows:

Adsorbed mass (ng/cm2) =

ଵ଻.଻ ௡

× Δf (Hz)

Where, n is the number of overtones


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2.2.7 Synthesis and Characterisation of PEG-based Hydrogels 2.2.7.1

PEG functionalization

Norbornene-functionalised PEG was prepared as described elsewhere12. Briefly, the 8-arm PEG-hydroxyl (Mw 10,000) was dissolved in dichloromethane (DCM) with 5× pyridine (with respect to hydroxyl concentration) and 5× 4-(dimethylamino) pyridine in one reaction vessel. In a separate vessel, 5× DCC (with respect to PEG hydroxyl groups) was reacted with 10× 5-norbornene-2-carboxylic acid at room temperature. The norbornene anhydride was then transferred to the PEG-hydroxyl solution using a cannula. The reaction mixture was allowed to proceed at room temperature under nitrogen overnight. Afterwards, the reaction was filtered and concentrated under rotary evaporation and precipitated (3x) in ice-cold diethyl ether. The filtered PEG-norbornene powder was then dissolved in water and lyophilized. PEG-norbornene was dialysed against water with molecular cut off of 5kDa for 3 days before being used.

2.2.7.2

Nuclear magnetic resonance

Quantitative 1H NMR (400 MHz) spectra for PEG-norbornene was acquired on a Bruker Av400X high-resolution NMR spectrometer. Freshly dialysed samples (dialysed for 3 days with molecular weight cut-off 3.5kDa to remove unreacted norbornene) were dissolved in chloroform prior to analysis. The degree of substitution of norbornene on PEG macromers was calculated from the integral of the methylene resonance from PEG (δ = 3.7ppm) and the double bond from norbornene (δ = 6.0 and 5.9 ppm), normalizing the number of contributing protons. 1HNMR spectra before and after conjugation of norbornene are shown in Supplementary figure S1. Integration of the peaks for PEG (labelled ‘1’) and norbornene (labelled ‘2’) confirmed that the degree of functionalisation of the PEG with norbornene was 88 %.



2.2.8 Hydrogel formation 8-arm norbornene functionalised PEG was cross-linked with 4-arm PEG-thiol in the presence of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photo-initiator under UV light (intensity of 180 mW/Cm-1) with exposure time of 3, 5 and 10s at room temperature. A stock solution of 20% w/v PEG-norbornene and 10% w/v PEG-thiol and 5mM LAP was prepared. The thiol to norbornene ratio was 1:1. LAP was added to a final concentration of 0.05% w/v.

PBS buffer was used to make up to the final desired PEG-norbornene

concentration. For PBP conjugated gels, peptides were added to the gel solution to make up 200µM. The concentration of LAP was kept at 0.05 %w/v. 2.2.9 Cryo-SEM imaging Hydrogel samples were cryogenically imaged using a Polaron LT7400 cryo prep chamber that was attached to a Philips XL30 Field Emission Scanning Electron microscope (FESEM). An accelerating voltage of 2kV was used for the images. High pressure freezing method was used to freeze the samples, as described by Aston et al.,21. The samples were sublimed (within the cryo prep chamber) for 45 minutes at -80oC before being placed into the SEM chamber at -190˚C. 2.2.10 Rheometry The shear elastic (storage) modulus (G′) of the PEG-based hydrogels were measured using a Discovery HR-3 hybrid rheometer (TA Instruments, New Castle, DE) with 8 mm diameter stainless steel flat top plate in oscillatory mode. Gels were prepared as discs of diameter of 8 mm and thickness 1mm and immersed in PBS for 24 hours prior to testing in the rheometer. A strain sweep from 0.1 to 100% was conducted at a frequency of ω0=1.0 Hz (6.3 rad s-1) at room temperature to determine the linear viscoelastic (LVE) region of fully hydrated cross-linked gel. Frequency sweeps from 0.01 to 100 Hz were conducted using a


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LVE strain amplitude determined in the strain sweep. A time sweep was performed on these gels using the values obtained from strain and frequency sweeps to accurately report the storage (elastic) modulus. Gels with different monomer concentrations and UV exposure times were tested in triplicate.

2.2.11 Extracellular matrix capture (cell-free and cell-loaded) in 3D hydrogels Cell-free: PBP-conjugated PEG-NRB/PEG-Thiol hydrogels and non-conjugated PEG-based hydrogels were incubated in fibronectin, collagen I and laminin I solution for two days to test the ability of PBPs to capture ECM proteins. Thereafter, all gels were incubated in water for 24 hours to allow un-bound ECM protein to leach out before staining for fibronectin, collagen I and laminin, as described below. Cell-loaded: hMSCs at 400,000 cell/ml were centrifuged to form a pellet. The supernatant was removed and non-cross-linked PBP-conjugated and non-conjugated PEG-NRB/PEG-Thiol macromere solutions were added to the pellet and mixed to make a homogenous suspension of cells in each polymer solution. Gel components and cells were then immediately cross-linked and the hydrogel discs incubated in culture media for up to 21 days. Secreted proteins within these gels at day 7, 14 and 21 were stained and imaged as detailed below. 2.2.12 Immunofluorescence staining in hydrogels 1mm×6mm gel disks were incubated in 1%BSA blocking solution overnight prior to incubating in primary antibody solution of a mouse monoclonal α-fibronectin antibody (Sigma Aldrich, F0916), a mouse monoclonal α-Collagen type 1 antibody (Sigma Aldrich, C2456), a mouse monoclonal α-Laminin antibody (Sigma Aldrich, L8271) for 18 hours. Then



the gels were washed with 1% BSA overnight before incubation in Anti-mouse IgG1 secondary antibody conjugated with Alexa Fluor 647 (Life Technologies, A21121), Hoechst 33342 (Invitrogen diluted 1:1000) and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific diluted 1:40) overnight to stain for ECM protein, nuclei and f-actin, respectively. 2.2.13 Imaging and analysis of ECM capture from supernatant (cell-free) and cellsecretions in 3D hydrogels The binding or capture of ECM proteins by protein binding peptides within the hydrogels was investigated using an Operetta (PerkinElmer) high content spinning disk confocal microscope with 10X objective. Fluorescence intensity was quantified using Harmony 4.1 software (PerkinElmer). Images of ECM proteins and hMSCs in a non-conjugated PEG-based hydrogel and PEG-PBP conjugated hydrogels were collected with an LSM 780 (Zeiss) confocal microscope using a 40X W 1.4 Korr M27 C-Apochromat objective. Excitation wavelengths were 405, 488 and 647 nm for Hoechst, Alexa Fluor 488 and Alexa Fluor 647 respectively. Images were analysed using Imaris® 8.4 software.

2.2.14 Statistics Statistics analysis was performed using Prism 7.0 (GraphPad Software Inc). Unless otherwise stated, data are presented as mean ± Standard Deviation, with p-values generated from unpaired t tests comparing all conditions versus control with a significance of p

Encoding Stem Cell-Secreted Extracellular Matrix Protein Capture in

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