alginate hydrogels open new scenario for articular tissue engineering applications

Accepted Manuscript Enhanced mechanical performances and bioactivity of cell laden-graphene oxide/ alginate hydrogels open new scenario for articular tissue engineering applications A. Marrella, A. Lagazzo, F. Barberis, T. Catelani, R. Quarto, S. Scaglione PII:

S0008-6223(17)30047-7

DOI:

10.1016/j.carbon.2017.01.037

Reference:

CARBON 11646

To appear in:

Carbon

Received Date: 17 August 2016 Revised Date:

16 December 2016

Accepted Date: 13 January 2017

Please cite this article as: A. Marrella, A. Lagazzo, F. Barberis, T. Catelani, R. Quarto, S. Scaglione, Enhanced mechanical performances and bioactivity of cell laden-graphene oxide/alginate hydrogels open new scenario for articular tissue engineering applications, Carbon (2017), doi: 10.1016/ j.carbon.2017.01.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Enhanced mechanical performances and bioactivity of cell laden-graphene oxide/alginate hydrogels open

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new scenario for articular tissue engineering

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applications

A. Marrella1,2, A. Lagazzo3, F. Barberis3, T.Catelani4, R. Quarto2, S. Scaglione1*

CNR - National Research Council of Italy, IEIIT Institute, Via De Marini 6, 16149 Genoa, Italy

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Department of Experimental Medicine, University of Genoa, Largo R. Benzi 10, 16145 Genoa,

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1

Italy

Department of Civil, Chemical and Environmental Engineering, University of Genova, p. J.F.

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Kennedy 1, 16129 Genoa, Italy

Italian Institute of Technology (IIT), Electron Microscopy Laboratory, Via Morego 30, 16163

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Genoa, Italy

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*Corresponding author. Tel. 0039 0106475206 E-mail: [email protected]

ABSTRACT

The development of novel 3D systems is crucial for engineering artificial tissues since the

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behavior of cells growth on 2D cell culture substrates does not accurately reflect that of the

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physiological microenvironment. In this regard, desirable 3D composites should offer tunable structural and functional properties to support appropriate cellular growth and biomechanical loads. In this work, we realized 3D alginate hydrogels functionalized with graphene oxide (GO) nanosheets for the creation of cell laden hybrid materials with proper mechanical properties for tissue engineering applications. We monitored the mechanical proprieties of 2 wt% GO/Alg

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hydrogels up to one month demonstrating a significant improvement of the compressive elastic modulus reaching values of 300KPa (6 times higher stiffness), which are close to those of articular tissues. This finding has been correlated to increased intermolecular hydrogen bonds

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over time between GO and Alg, observed through FT-IR analysis. Interestingly, we show that

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3D GO/Alg hydrogels trigger cellular activity in vitro, as demonstrated by the statistically significant improvement of the viability of fibroblasts encapsulated in GO/Alg hydrogels and by the absence of cytotoxicity of suspended GO flakes. All these findings indicate that GO/Alg hydrogel is a promising material for articular tissue engineering, where biomechanical requirements are crucial.

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1. Introduction

Tissue engineering aims to overcome the limitations of traditional clinical practice, developing

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biological substitutes to restore, maintain or improve functions of a tissue or whole organs [1]. To reach this goal, cells should interact with implantable three-dimensional scaffolds, which provide structural support while fostering new tissue formation [2]. The development of 3D

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scaffolds is thus a key point for tissue engineering: their mechanical stiffness, chemical composition, biodegradability and internal architecture must be properly designed to realize

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biomaterials able to guide the correct tissue regeneration. In this context, hydrogels have been widely proposed as biological substitutes thanks to their structural similarity to the macromolecular-based components of the native extracellular matrix (ECM); thus, they have the potential to direct migration, growth and organization of the cells during tissue regeneration [3,

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4]. The structure of the hydrogels, which are networks of hydrophilic biopolymers, is stabilized either by chemical crosslinking (covalent and ionic), or physical crosslinking (entanglements, crystallites, and hydrogen bonds). These materials can be degraded and dissolved by the

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biological environment creating micro-pores where host cells may penetrate and proliferate; alternatively, they can be directly designed for some biomedical applications with high rate of

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total porosity for accommodating living cells, or as matrixes for encapsulation and delivery of cells as well as bioactive molecules [5, 6]. Both synthetic and natural polymers have been commonly used for realizing 3D hydrogels: the first class (e.g. poly-vinyl alcohol, poly-ethylene glycol, polyacrylamide, and polypeptide) are generally easily processable and exhibit good mechanical properties; however, they typically display and slow degradation rate [7-10]. Natural polymers (e.g. elastin, collagen, gelatin, fibrin,

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alginate, hyaluronic acid, chitosan) are chosen for their good biocompatibility; among them, alginate (Alg), which is a polysaccharide extracted from brown seaweeds, displays very good scaffold-forming properties since it can be easily processed in creating different forms such as

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porous sponges, microcapsules or microspheres [11, 12]. Due to its outstanding properties in terms of biodegradability, non toxicity, non immunogenicity and chelating ability, alginate has been widely used in a broad range of biomedical applications both as a biomaterial, including

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delivery system for tissue repair and regeneration [13-17].

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wound dressings, dental impression materials, cartilage and bone tissue engineering, and as cells

However, many natural polymers are characterized by an overall mechanical weakness which limits their extended use in preclinical/clinical applications [18, 19]; thus it becomes mandatory to mechanically reinforce these matrices for providing structural properties similar to the specific target tissues. Alginate hydrogels are soft in nature; their compression modulus can range from 1

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to 1000 kPa with a shear modulus from 0.02 to 40 kPa [20]. This range is found to be much less than ECM materials, such as collagen or elastin.

Mechanical properties are highly dependent on the source of the polymer, method of processing,

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concentration and type of crosslinking ion and gelling environment. A common technique used

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to improve stiffness of natural polymers is to increase the cross-linking density [21], although it often induces to a lower permeability and a reduced hydration upon swelling [22]. Alternatively, synthetic nanomaterials (i.e. nanoparticles, nanosheets, nanofibers) have emerged as mechanical reinforcing agents [23, 24]. In particular, they may both better emulate the native microenvironment, thus fostering the cellular interaction with the artificial substrate, and strengthen the 3D network of the hydrogels, through a chemico-physical functionalization of the hydrogels [23, 25, 26].

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The introduction of nano-reinforcements into alginate hydrogels has been performed by several studies, where nanomaterials such as nano silica, hydroxyapatite, carbon nanotubes (CNTs) were added to the polymeric matrix to enhance the final outcome [27, 30].

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Although great results in terms of an enhancement of the mechanical strength of the hydrogels were obtained through the additions of CNTs, they often need a chemical functionalization to being dispersed in organic solvents and a purification to eliminate the impurities introduced

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during their fabrication process [30, 32]. In fact, recently cytotoxic effects have been observed and attributed to the incomplete removal of metal catalysts used to prepare CNTs [33, 34].

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Moreover, CNTs tend to aggregate because of their hydrophobicity, resulting in a heterogeneous interaction with cellular components [35].

Graphene and its derivatives have recently emerged as promising alternative nanomaterials due to their unique mechanical, physical, chemical proprieties [12]. Graphene/graphene oxide (GO)

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based material has become one of the most interesting materials in many different fields including biomedicine [36]. GO is an atomical thin sheet with very large specific surface area and a large number of functional groups (e.g. hydroxyl, epoxide, and carbonyl) bound on the

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surface. GO is more widely used than graphene in biomedical applications due to the presence of carboxylic, epoxy and hydroxide groups, which allow for wide range of reactions and

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functionalization [36]. GO shows excellent mechanical properties (high elasticity, strength, flexibility) and it can be potentially used as reinforcement in hydrogels. Recent studies have explored the effects of the introduction of low concentrations of GO in polymeric matrix, such as PVA-based films [37], gelatin based materials [38], chitosan scaffolds [39]: in these studies the mechanical strengths of the hybrid materials appeared significantly enhanced without affecting the polymeric cytocompatibility. Other preliminary works suggest an improvement of the

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functional and mechanical proprieties of alginate/GO composites, prepared either by wetspinning or as films [40, 41]. However, many efforts still have to be done to realize 3D clinically

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relevant cell laden Alg based hydrogels suitable for tissue engineering applications. For these reasons, our aim is to combine GO with Alg in order to realize novel 3D hydrogels with tunable mechanical properties and to evaluate the effects of GO inclusion on the structural

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and biological features in a 3D environment, providing the basis towards alternative materials for regenerative medicine applications.

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The combination of a polysaccharide and nanomaterials leads to the formation of a hybrid material, which can provide a feasible and effective method to modulate the physico-chemical and biological properties of the hydrogel. The synergistic effect of multiple structures often produces new hydrogels that can exhibit a novel structure organization and new properties that

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greatly expand the scope of their application in several areas of tissue engineering. Here, following an extensive physico-chemical characterization of the 3D GO/Alg based matrixes, the study of cell-material interactions has been performed using fibroblast cell line.

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Challenges in fabrication of cell-laden 3D GO/Alg hydrogels are: (a) the generation of constructs with clinically relevant size for clinical applications; (b) the realization of constructs with

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adequate mechanical performances; (c) the stabilization of the cell–hydrogel constructs without adverse effects on cell viability.

2. Experimental 2.1 Fabrication of GO/alginate hydrogels

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Graphene oxide flakes (Sigma Aldrich, Product No. 763713) aqueous suspension (1mg/ml) was prepared by ultrasonic treatment (FALC Instruments, LBS1) for 3 hours suspending graphene oxide in distilled water. The homogeneity of the GO dispersion was assessed by optical

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microscopy. Specifically, a small volume (0,05 ml) of the dispersion was placed onto a glass microscope slide and covered with a cover slip to ensure appropriate imaging thickness and to prevent drying. Images of the dispersions were obtained with an optical microscope (Nikon

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H550L).

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Physiologic solution was prepared dissolving NaCl in deionized water (0,9% w/v). Alginate (Manugel GMB, FMC Biopolymer) was dissolved in physiologic solution at different concentrations to obtain an alginate final concentration about 2% w/v for all the samples realized with different contents of GO. Composite GO/Alg mixtures were produced by drop wise GO suspension to the sodium alginate solutions, to obtain different concentrations of GO (0, 0.5, 2

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wt% GO/Alg). The resulting solutions were constantly stirred for about 30 min using a magnetic stirrer. GO/Alg mixtures were then poured in disk-shaped agarose molds. In brief, an agarose solution (1% w/v) was prepared dissolving agarose powder (Sigma Aldrich, Product No. A1296)

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in 0,1M CaCl2 solution. The solution was stirred and boiled and then it gelled cooling at ambient

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temperature. Cylindrical holes were realized inside the agarose gels with 5 mm punches and the GO/Alg mixtures were poured inside them. The composite mixtures were chemically crosslinked for 2 hour at 37 °C, through the calcium ions radial diffusion from the surrounding agarose gels.

2.2 Cell laden GO/Alg hydrogels

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Mouse fibroblast cell line (NIH-3T3) was expanded in a Dulbecco’s Modified Eagle’s Medium (DMEM)

supplemented

with

10%

fetal

bovine

serum

(FBS)

and

1%

(v/v)

penicillin/streptomycin (complete medium). The culture media was changed twice a week. When

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cells became confluent and reached the required number, 3T3’s were enzymatically detached with 0,05 % trypsin and then counted. Cells were suspended in GO/Alg mixtures (1,25 millions/ml) and all it poured in the agarose molds. The solutions were chemically cross-linked

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for 2 hour in incubator at 37 °C. Then, the hydrogels were cultured in cell culture well-plates up to 28 days using complete medium and CaCl2 (5mM) in an incubator in an atmosphere of 5%

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CO2 respectively, allowing gas exchange in the reservoir at 37 °C. 2.3 Viability tests

The viability of cells embedded in hydrogels constructs (0, 0,5 wt%, 2 wt% GO/ Alg) was

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examined by a live/dead assay (Sigma Aldrich) on day 1(T1), 4(T4), 1 week (T7), 2 weeks (T14). Briefly, samples were washed with phosphate buffered saline (PBS, Sigma-Aldrich), incubated in 2mM calcein AM (living cells) and 4mM EthD-1 (dead cells) in PBS for 15 min at

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37 °C, then washed with PBS again. Cell-laden hydrogels were cut in half and observed under optical fluorescence microscope both in the cutting and on the outer sections. No significant

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differences were observed in terms of cell distribution and viability. Staining was observed by filtering for calcein at 498–525 nm, and for ethidium homodimer-1 at 553–765 nm. Viable and dead cells were counted using Image J software (National Institutes of Health, Bethesda, MD, USA).

2.4 Proliferation tests

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Cell proliferation of cells embedded in hydrogels constructs (0, 0,5 wt%, 2 wt% GO/ Alg) was assessed by using a cell viability reagent (Presto Blue Assay, Thermo Fisher Scientific). In brief, after one (T1), seven (T7) and ten days (T10) the assay was added to the culture medium at a

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concentration of 10% v/v to each well, as indicated. Samples were then incubated at 37 ◦C for 45 minutes in the dark. The supernatant was then removed and its absorbance was quantified at 570 and 600 nm by spectrophotometry. All the experiments were performed in triplicates. The levels

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2.5 h-MSCs culture under chondrogenic conditions

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of cell proliferation were expressed as number of cell doublings over time.

For the biological validation of the developed hydrogels, human mesenchymal stem cells (MSC) were used. Bone marrow derived MSCs were obtained by healthy donors, expanded in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum

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(FBS), 1% (v/v) penicillin/streptomycin (complete medium) and 5 ng/ml fibroblast growing factor-2 (FGF-2) as previously reported [42]. MSCs were expanded in vitro until passage 2 and medium changes were accomplished every 3 days. When reached confluence, MSCs were

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enzymatically detached with 0,05 % trypsin, counted, suspended in GO/Alg mixtures (1,25 millions/ml) and embedded in the hydrogels (Alg and 2 wt% GO/Alg).

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Cell-laden hydrogels were cultured in a defined chondrogenic medium for 3 weeks, as previously reported [43]. Briefly, it consists of complete medium supplemented with 5 mM CaCl2, 10-6 M bovine insulin, 8x10-8M apo-transferrin, 8x10-8M bovine serum albumin, 4x10-6M linoleic acid, 10-3M sodium pyruvate, 5 mg/ml ascorbic acid, 10−7M dexamethasone and 10 ng/ml TGF-β3. All the reagents were purchased from Sigma Aldrich. Culture medium was changed three times a week. After 3 weeks, cell-laden hydrogels were processed for histological analysis. Generated constructs were fixed in 4% buffered formalin for 2 hours, then paraffin-embedded, sectioned (5

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micron) at different levels and stained with Alcian Blue and Safranin O red staining. The histological sections were observed and evaluated by optical microscopy in transmitted light (Nikon H550L).

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2.6 Structural and chemical characterization of GO/alginate hydrogels

The spatial distribution of GO in the GO/Alg hydrogels was evaluated thorough histological

and examined with a light microscope (Nikon H550L).

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analysis. Hydrogels were paraffin embedded, sectioned (5 micron), dewaxed, stained with eosin

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FT-IR skeletal spectra of dried hydrogels were recorded after dilution of the ground sample powder with KBr (Aldrich, FT-IR grade) (1%, w/w) by a Nicolet Nexus Fourier transform instrument (OMNIC™ software, DTGS detector). FTIR analyses were performed over samples at different times of in vitro cell culture to evaluate potential chemical modifications during the

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in vitro cell culture for 2 wt% GO/Alg and Alg hydrogels. Analyses were performed after 4 days (T4) and 28 days (T28) of cell culture for both functionalized and not alginate hydrogels. 2.7 TEM analysis of GO/alginate hydrogels

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Small blocks of GO/alginate hydrogels were fixed for 2 hours in 2% glutaraldehyde in

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physiological solution buffered with 5mM CaCl2. Subsequently, the sample were post-fixed for 2 hours in 1% osmium tetroxide in the same buffer and stained overnight in a mixture of 1% uranyl acetate in aqueous solution. The samples were then slowly dehydrated in an increasing series of alcohols and then cleared in propylene oxide. Finally the samples were gradually infiltrated in Spurr resin (SPI-Chem). After polymerizations at 65 C for 48 hours, the samples were cut in 70 nm thick ultra-thin sections using a Leica EM UC6 ultra-microtome. TEM images were collected with a Jeol JEM 1011 (Jeol, Japan) transmission electron microscope (Electron

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Microscopy Lab. – Nanochemistry Dept., Istituto Italiano di Tecnologia) operating at an acceleration voltage of 100 kV, and recorded with a 11 Mp fiber optical charge-coupled device

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(CCD) camera (Gatan Orius SC-1000). 2.8 Mechanical Analysis

Measurement of mechanical properties (compression elastic modulus) of the Alg based

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hydrogels was carried out using a home-made loading instrument consisting of a cylindrical indenter with a diameter of 5 mm, which is pushed down on a vertical cylindrical sample of 5

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mm in diameter and 4.5-mm in height. Samples were swollen in deionized water before test and were pre-loaded with 20% of strain. Storage and loss modulus of Alg based hydrogels either functionalized or not with GO (2 wt%) were assessed up to 28 days of culture (with and w/o cells) at different time points with stimulation frequencies ranging from 0,5 Hz to 10 Hz.

deviation. 2.9 Statistical analysis

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Measurements were recorded in triplicates and the results were expressed as mean and standard

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Experiments were all performed in triplicate. Data about cells viability and mechanical

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proprieties are reported as mean ±standard deviation. Data were analyzed by Mann-Whitney U test, n=9. Statistical significance was set at p<0.05. 3. Results and Discussion

3.1 Three-dimensional GO/Alg hydrogel realization A simple sequential fabrication process was designed (Figure 1A) to produce 3D composite GO/alginate hydrogels with clinically relevant size (3 mm of height and 5 mm of diameter).

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Commercially available graphene oxide was produced through oxidation/exfoliation of graphite powder via the modified Hummer’s method. Go nanoflakes displayed mean lateral size of 6.28 µm ±0,16 and mean thickness of 4 nm±0,28. The exfoliated GO nanosheets were readily

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dispersed in deionized water with ultrasonic treatment and formed a suspension that is stable for several months with no precipitation. An agarose mold was adopted to create uniform diskshaped GO/alginate hydrogels; GO/Alg mixtures (with and without cells embedded) were cast in

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the Ca2+ ion-releasing agarose molds and radially cross-linked by diffusion of Ca2+ ions from the

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molds.

Negative ions present both in Alg and GO solutions allowed to produce a homogeneous and stable suspension [41]. Being GO flakes brown, the GO flake conjugation to alginate scaffolds changed the hydrogel color from white to brown according to the GO concentration (Figure 1B). No evidence of aggregation was observed for each GO/Alg hydrogel composite, suggesting a

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spatially homogeneous distribution of GO throughout the hydrogels. To confirm the dispersion of the GOs within hydrogels, histological analysis of 3D scaffolds was performed: eosins

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staining revealed homogeneous distribution of GO flakes throughout the volume (Figure 1C).

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Figure 1. Photograph of GO dispersion in DI water (1mg/ml) (Panel A); optical microscope image of a typical stable GO dispersion in water (Panel B), schematic representation of the process of GO/Alg hydrogels realization (Panel C); photos (panel D) and histological eosin-stained images (Panel E) of GO/Alg hydrogels with different GO contents. 3.2 Cells viability, proliferation and hMSCs differentiation onto Alg and GO/Alg hydrogels To investigate whether the cell-material interactions are compromised by the presence of GO in

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the hydrogels, the viability and growth of encapsulating 3T3 fibroblasts were analyzed. The results were compared between Alg and GO/Alg, using different GO concentrations for

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evaluating possible cytotoxicity effects. We qualitatively monitored the cells viability by staining them with calcein/ethidium and observing them under the confocal microscope up to two weeks (Figure 2). Biological proprieties are, in fact, the most important features that determine the scaffolds usefulness in tissue engineering, or more in general, in biomedical applications.

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Cell viability tests showed that the presence of graphene oxide does not decrease cell viability, confirming absence of toxicity, at least up to 2% wt of GO; interestingly, cell viability was higher increasing the concentration of GO, suggesting a positive effect of graphene on biological

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activity in vitro (e.g. cellular adhesion and proliferation). For all time points the cell viability was

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statistically higher in hydrogels functionalized with GO, while there was no significant difference between 0.5 wt% and 2 wt% GO/Alg (Figure 3). The number of 3T3s cells doublings within the hydrogel reveal a good proliferation rate, definitively not affected by the introduction of GO, at least up to 2% wt of GO (Table 1). Based on these findings, the following chemico-physical investigations have been carried out comparing Alg- and 2 wt% GO/Alg- hydrogels.

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Figure 2. Cells viability after different times of cell culture for hydrogels with different GO content (Green spots are alive cells, red spots are dead ones). Scale bar is 50 µm.

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Figure 3. Quantitative analysis of cell viability after different times of cell laden hydrogels with different GO content (p<0.005). N° Doublings after 7 days

N° Doublings after 10 days

2.94 ±0.30

3.25 ±0.29

0.5 wt% GO/Alg

2.61 ±0.40

2.82 ±0.48

2 wt% GO/Alg

2.73 ±0.73

2.78 ±0.77

Alg

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Table 1. Number of doublings of 3T3s embedded in hydrogels with different GO content, after 7 days and 10 days of cell culture (mean and standard error).

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These results indicate that the presence of GO in alginate matrixes is not harmful for the embedded fibroblasts in terms of viability, but enhanced it. Although the mechanisms regulating this behavior have not been yet elucidated, the observed increase in cellular viability could be possibly due to the stronger cell adhesion onto GO/Alg hydrogels, as also reported by similar

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works related to carbon-based nano-materials addition [44, 45]. The presence of nanostructures

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may in fact alter the surface hydrophilic/hydrophobic character, its stiffness and nano-roughness, which are all important factors playing a significant role in cell adhesion and culture. After 3 weeks, hMSCs cultured within alginate based hydrogels in presence of chondrogenic medium organized a tissue structure with a clear alcianophilic extracellular matrix containing cells encapsulated in their lacunae, showing a chondroid like morphology (Figure 4). A strong

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diffuse staining for Alcian Blue was in particular observed in hydrogels functionalized with graphene oxide, confirming the presence of a chondrogenic matrix rich of glycosaminoglycans (GAG).

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These results demonstrate that the GO/Alg hydrogel is permissive to selective cellular differentiation and chondrogenic matrix deposition.

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Further studies could be carried out to evaluate the functional performances of these hydrogels implanted in small animal models, in orthotopic site.

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Figure 4. Safranin O Red/Alcian Blue staining of Alg hydrogel (panel A) and 2 wt% GO/Alg hydrogel (panel B) cultured with hMSCs after 3 weeks (Scale bar = 20 µm).

3.3 Three-dimensional GO/Alg hydrogel characterization

FT-IR analyses were performed to investigate the absorption bands spectrum of Alg and GO/Alg composite hydrogels (Figure 5). The spectra were similar to those previously presented in

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literature [46, 47]. In particular, in the Alg spectrum the peaks at 1000-1100 cm-1 are attributed to the stretching vibration of C-C and C-O bond groups, respectively [47], while the peaks in the range 1421-1615 cm-1 correspond to symmetric and anti-symmetric COO− stretching vibration

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of carboxylate group. The introduction of GO within the Alg hydrogel is detectable above 3000 cm-1, where surface OH groups are visible (Figure 5A-B). Interestingly, some features are

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detected changed in shape and position comparing pure Alg spectrum and GO/Alg spectrum, indicating a chemical interaction between the two components (Figure 5A-B). In particular, when GO was added, the peaks between 1100 and 1700 cm−1 appeared shifted; moreover, the dominant absorption at 3447-4000 cm−1 assigned to OH stretching vibrations drastically broadened, especially after 28 days, thus suggesting an increased interaction over time among surface oxygenated groups of GO and Alg through intermolecular hydrogen bonds; this achieved

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electrostatic attraction between the two components, also shown in bi-dimensional systems (i.e. films), is responsible of an enhanced GO/Alg interfacial adhesion [40].

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The FT-IR analysis of cell laden GO/Alg hydrogels at different time points was also performed to evaluate chemical stability of the materials over time (Figure 5C-D). Absorption bands at 3000-3500 cm-1 of Alg and GO/Alg hydrogels FT-IR spectrum displayed significant changes in

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shape: in both materials, peaks appeared sharper after 28 days, suggesting a more ordered

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structure of hydrogels over time.

Figure 5. FTIR spectra of alginate hydrogels with and without GO (Panels A, B) and at different times of cell culture (Panels C, D).

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TEM analysis was performed to analyze the GO sheets organization at submicron level within the Alg matrixes (Figure 6). Both at early and prolonged time points, the GO sheets were mainly

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clustered in aggregates of various shape ranging in size from 0.5 to 5 microns, with a homogeneous spatial distribution within the hydrogels and no preferential spatial orientation (Fig. 6a-b). Moreover, at higher magnification we didn’t observe any relevant nanofibers organization changes over time (Fig. 6c-d): in particular, the size of GO aggregates seems to remain constant suggesting that there’s no aggregation nor sheets preferential ordering over time. Regarding cell distribution and interaction with GO in the Alg matrix, TEM analysis showed that cells are present both isolated and in small aggregates of 5-10 cells (data not shown), interacting

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with GO flakes (Fig. 6e-f). Cells close to GO aggregates exhibited a regular and healthy nuclear and cyplasmatic membrane, avoiding any possible risk of cytotoxicity by GO, at least at these concentration and incubation times. Moreover, we observed in some cases some membrane

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features suggesting a initial GO sheets incorporation by the cells, suggesting good bio-

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compatibility.

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3.4 Mechanical proprieties of Alg and GO/Alg hydrogels

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Figure 6. TEM images of graphene oxide sheets size and distribution within alginate matrix after 4 days (panels a, c, e) and 28 days (panels b, d, f) of cell culture. Images were acquired at low (panels a-b) and high (panels c-d) magnifications; cell-GO interaction was also investigated (panels e-f).

The mechanical properties of 3D hybrid hydrogels were evaluated by dynamical mechanical

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analysis (DMA), with a frequency ranging 1-10 Hz. DMA is a non-destructive tool that permit to obtain information both on the elastic and on the viscous component of the material, thus

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particularly adequate for characterizing soft biomaterials. The hydrogels functionalized with GO displayed an initial compressive Elastic Modulus about 3 fold higher than the Alg control (Figure 7). After 1 day of cell culture, an overall decrease of the compressive modulus was observed for the all hydrogels; this result is consistent with the water uptake ability of polysaccharides and the

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partial alginates degradation, decreasing the stiffness of the matrixes [5, 48]. Interestingly, a dramatic enhancement of the stiffness was observed from one week up to four weeks of cells culture only for GO/Alg functionalized hydrogels (Figure 7). To decouple the possible biological

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and biochemical effects, the Elastic Modulus was measured at the same time points in both cell laden- and unloaded- hydrogels; no significant differences were observed, thus confirming that

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the cells activity does not drastically affect this process.

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Figure 7. Elastic Modulus of alginate hydrogels with and without graphene oxide (2 wt%) up to one month of in vitro cell culture.

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The variations of the compressive Dynamic Elastic Modulus values, also known as storage

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modulus E’, give the information about the change in mechanical properties of the materials and it can be connected with the degree of crosslink of the polymeric compound. Considering the theory of rubber elasticity, the crosslinking density (νe) may be determined by the following equation: (1)

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E' = 3 νe RT

Where E' is the storage modulus of the cross-linked polymer in the rubbery plateau region above Tm (E'rubber), R is the gas constant (8.314 J·K−1·mol−1) and T is the absolute temperature (K)

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[49]. The measurements over time demonstrate a significant enhancement of E’ in GO/Alg samples if compared with that of Alg hydrogels, validating that the introduction of GO leads to a

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stiffer material, through new chemical cross-links in the hydrogel and the action of long range capillary forces among the GO particle and the alginate matrix (Figure 6) [50]. This result is also in agreement with the FT-IR analysis discussed above. Moreover, analyzing the variations of the storage modulus E’ over frequencies, we may observe a stable behavior of Alg samples from 1 to 10 Hz during one month of observation, confirming a compressive elastic modulus of about 50KPa (Figure 8A). On the other side, GO/Alg matrixes

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exhibited after 1 week up to 4 weeks of culture a significant higher E’ for all frequencies, with

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the maximum values measured at low frequencies (Figure 8B).

Figure 8. Analysis of the variation of storage modulus over frequencies at different times of culture (time 0 (t0), after 1 day (t1), 7 days (t7) and 28 days (t28)) for Alg (Panel A) and GO/Alg hydrogels (Panel B).

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The damping properties of the materials were also investigated by monitoring the variation of the

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loss factor (tanδ) with frequency (Figure 9), obtained as ratio loss modulus (E”) over storage modulus (E’) [51]. A light and continuous decrease with the frequency in the Alg samples can be observed (Panel B), indicating that the materials become less viscous and more elastic. Since this parameter is connected with the capacity of the material of storing energy when it is subjected to a stress, a lowering of the loss factor is sign of a tendency towards a solid-like behavior. This was also in agreement with previous studies, showing that CaCl2 - cross-linked alginate matrixes become more elastic as the frequency increases [52].

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Interestingly, starting from a week, 3D GO/Alg hydrogels displayed a more elastic behaviour of at physiological frequencies (in the range of 1Hz) despite a viscoelastic outcome at higher

dissipate more energy become helpful (Panel A).

= tan δ, where the capacity to

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frequencies (revealed by higher loss factor

This biomechanical behaviour becomes very interesting for future application of GO/Alg

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hydrogels in tissue engineering. It is reported that the proper range of elastic modulus for articular tissue is in the range of 300–700 kPa [53]. Therefore, it seems that 2 wt% GO/Alg

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hydrogels can be suitable for using in cartilage tissue engineering regarding the mechanical properties. Moreover, compressive stiffness, as well permeability, is highly correlated with water content: as the water content increases, material becomes less stiff and more permeable [54, 55]. Therefore, a fine-tuning between mechanical and chemico-physical features shall has been done

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to balance structural and biological requirements of engineered tissues.

Figure 9. Analysis of the variations of the loss factor with frequency for GO/Alg (Panel A) and Alg hydrogels (Panel B). 4. Conclusions

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This study describes for the first time the production and characterization of 3D cell laden hybrid hydrogels based on a polysaccharide, Alg, and graphene oxide, GO. These nanofunctionalized hydrogels were undergone to an extensive physicochemical characterization to

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highlight the effect of GO on biocompatibility and biomechanical outcome of the composites for their possible use in biomedical applications. If Alg hydrogels lack the tensile and compressive strengths to resist functional loading, they will collapse in vivo. To avoid these complications

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that limit their use, structural modifications are needed to reinforce the alginates.

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In general, our results confirm (i) the absence of cytotoxicity of suspended GO flakes and the improved viability of fibroblasts encapsulated in GO/Alg hydrogels; (ii) a significant enhancement of compressive stiffness of GO/Alg hydrogels reaching 300KPa, comparable with articular tissue values, mostly due to new chemical links between the biopolymer and the GOs.

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We thus demonstrate that GO may act as both an effective reinforcement filler and a biological activator in hydrophilic alginates, offering the biopolymer–GO nanocomposites great potential to be further developed in biomedical fields. In particular, we have validated that the enhanced

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stiffness by GO incorporation within hydrogels does not affect the favorable characteristics of pure alginate such as bioactivity, making the 3D cell laden GO/Alg construct a promising hybrid

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materials for tissue engineering applications. Further studies are currently being carried out in order to evaluate the compatibility of this matrix with other types of cells, both in vitro and in vivo.

Acknowledgment

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The authors want to thank Prof. Finocchio for FTIR analysis, R. Marotta for TEM analysis and The Italian Ministry for Education, University and Research for funding.

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