Chondroinductive alginate-based hydrogels having graphene oxide for 3D printed scaffold fabrication

Subscriber access provided by ACCESO PROVISTO POR BEIC-CONICYT 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. Biological and Medical Applications of Materials and Interfaces Chondroinductive alginate-based hydrogels having graphene oxide for 3D printed scaffolds fabrication Felipe Andres Olate-Moya, Lukas Arens, Manfred Wilhelm, Miguel Angel Mateos-Timoneda, Elisabeth Engel, and Humberto Palza ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b22062 • Publication Date (Web): 07 Jan 2020 Downloaded from pubs.acs.org on January 8, 2020 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. 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Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts. 1 Chondroinductive alginate-based hydrogels having graphene oxide for 3D printed scaffolds fabrication Felipe Olate-Moya,*, 1 Lukas Arens,2 Manfred Wilhelm,2 Miguel Angel Mateos-Timoneda,3, 4, 5 Elisabeth Engel3, 4, 5 and Humberto Palza*, 1,6 1. Departamento de Ingeniería Química, Biotecnología y Materiales, Facultad de Ciencias Físicas y Matemáticas, Universidad de Chile, Beauchef 851, 8370456, Santiago, Chile. 2. Institute for Technical Chemistry and Polymer Chemistry (ITCP), Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76131, Karlsruhe, Germany. 3. Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Baldiri Reixac 10-12, 08028, Barcelona, Spain. 4. CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Monforte de Lemos, 3-5, 28029, Madrid, Spain. 5. Department of Materials Science, EEBE, Technical University of Catalonia (UPC), d'Eduard Maristany 16, 08019, Barcelona, Spain. 6. Millennium Nuclei in Soft Smart Mechanical Metamaterials, Beauchef 851, 8370456, Santiago, Chile. * To whom correspondence should be addressed. E-mail: [email protected] (F.O.-M.), [email protected] (H.P.); tel.: +56229780795 Page 1 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 2 KEYWORDS. 3D printing, graphene oxide, liquid crystals, hydrogels, chondrogenesis. ABSTRACT. Scaffolds based on bioconjugated hydrogels are attractive for tissue engineering because they can partly mimic human tissue characteristics. For example, they can further increase their bioactivity with cells. However, most of the hydrogels present problems related with their processability, consequently limiting their use in 3D printing to produce tailor-made scaffolds. The goal of this work is to develop bioconjugated hydrogel nanocomposite inks for 3D printed scaffold fabrication through a micro-extrusion process having improved both biocompatibility and processability. The hydrogel is based on a photocrosslinkable alginate bioconjugated with both, gelatin and chondroitin sulfate, in order to mimic cartilage extracellular matrix, while the nanofiller is based on graphene oxide to enhance the printability and cell proliferation. Our results show that the incorporation of graphene oxide into the hydrogel inks considerably improved the shape fidelity and resolution of 3D printed scaffolds, due to a faster viscosity recovery post extrusion of the ink. Moreover, the nanocomposite inks produce anisotropic threads after the 3D printing process, due to the templating of the graphene oxide liquid crystal. The in vitro proliferation assay of human adipose tissue derived mesenchymal stem cells (hADMSCs) shows that bioconjugated scaffolds present higher cell proliferation than pure alginate, with the nanocomposites presenting the highest values at long times. Live/Dead assay otherwise displays full viability of hADMSCs adhered on the different scaffolds at day 7. Noteworthy, the scaffolds produced with nanocomposite hydrogel inks were able to guide the cell proliferation following the direction of the 3D printed threads. In addition, the bioconjugate alginate hydrogel matrix induced chondrogenic differentiation without exogenous pro-chondrogenesis factors as concluded from immunostaining after 28 days of culture. This high cytocompatibility and chondroinductive effect toward hADMSCs, together with the improved printability and anisotropic structures, makes this Page 2 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 3 nanocomposite hydrogel inks a promising candidate for cartilage tissue engineering based on 3D printing. INTRODUCTION Additive manufacturing such as 3D printing is an emerging tool for biomedical applications in regenerative therapies.1 Particularly 3D printed scaffolding is a versatile method to obtain tailormade structures for tissue engineering and 3D cell culture.2 Among different biomaterials used for scaffold fabrication, hydrogels based on natural polymers have outstanding characteristics such as high water content, porosity, softness, biocompatibility and bioactivity.2,3 In addition, the properties and characteristics of these biopolymers can be modified through physical/covalent crosslinking or bioconjugation.4 From the 3D printing perspective, scaffolds based on alginate (ALG) have been widely studied due to its ability to change quickly from a viscous fluid state (an ALG solution) to a more-rigid hydrogel, by an ionotropic crosslinking process between α-Dglucuronic acid residues from the polymer chains and divalent cations.5 From the biological perspective, the inherent lack of proper biocompatibility and bioactivity of ALG has led to the strategy to combine this biopolymer with bioactive hydrogels such as gelatin (GEL) or its derivatives for biomedical applications.6 The purpose of this bioconjugation is to mimic the composition, microstructure, and mechanical properties of the extracellular matrix (ECM) of a determined tissue.7 Gelatin, particularly the photocrosslinkable derivative methacryloyl gelatin (GEL-MA), is often used for this purpose and for 3D printing.8 Gelatin is a polypeptide of animal origin, obtained from hydrolysis of collagen that is found in the ECM of most animal tissues. Scaffolds based on GEL have shown high cell viability, proliferation and cell attachment, due to the RGD amino acid sequence existing in its polymeric chains.9 For instance, scaffolds fabricated Page 3 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 4 from blends of ALG/GEL displayed enhanced cell proliferation compared with pure ALG scaffolds.10 Therefore, GEL and its derivatives appear as an easy and economic method of bioconjugation.9,11 Among the different potential applications of 3D printed hydrogel scaffolds, several studies have been focused on cartilage tissue engineering during the last decade.12 Cartilage is a tissue formed by cells called chondrocytes. It is characterized by a lacking of vascularization of its ECM and a poor regenerative capacity, so the injuries on this kind of tissue are difficult to treat.13 The ECM of cartilage (based on articular cartilage) consists of about 65-80 % of water, which is in the same range as hydrogels. Considering the dry weight, the principal components of cartilage are collagen (up about 60 %) and proteoglycans (30-40 %) consisting of proteins covalently bonded to glycosaminoglycans (GAGs).14 These GAGs are highly negatively charged polysaccharides consisting of a disaccharide repeating unit with an amino sugar moiety bonded to an uronic sugar o galactose moiety.15 With the aim of mimicking the ECM of cartilage tissues, previous evidence reports the use of GAGs as part of a hydrogel network in scaffolds.16 Among the different GAGs usually employed for these hydrogel scaffolds, hyaluronic acid, heparan and chondroitin sulfate (CS) stand out.17 From these three biopolymers, CS has great potential for cartilage tissue engineering because of its chondrogenic properties and low costs.18 CS is found in the EMC of cartilage covalently linked to protein forming the proteoglycan (PG) aggrecan, the principal PG constituting this type of tissue. Aggrecan has the ability to interact with hyaluronic acid chains forming PGs aggregates, which act like a hydrogel structure, able to retain water molecules maintaining the hydration of the cartilage ECM.15 Scaffolds based on CS can be formed either by mixing it with other polymer or by crosslinking it covalently with other polymeric chains to form the hydrogel network.19–21 From the regenerative therapy point of view, engineered chondral tissue Page 4 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 5 employing human mesenchymal stem cells (hMSCs) for chondrocyte phenotype differentiation, is an attractive option for the treatment of cartilage injuries.22 Therefore, GEL/CS-bioconjugated hydrogel scaffolds having hMSCs seem to be a proper approach for the design of biomaterials for cartilage tissue engineering.23,24 However, despite the good behaviours of ALG, GEL and CS for cartilage regeneration, scaffolds based on covalently bonded ALG-GEL-CS ternary hydrogels and manufactured by 3D printing has not yet been studied. 3D printing of scaffolds based on hydrogels has a series of complications related to its poor mechanical properties and low processability.25 These difficulties have motivated the development of reinforced hydrogels with nanomaterials like metallic nanoparticles, hydroxyapatite , clay and carbon-based nanomaterials.26 These nanocomposites do not only improve the hydrogel mechanical behaviour but also their rheological properties, usually showing additional attributes such as optical properties, anisotropic characteristics and bioactivity.27 One of the most interesting nanoparticle for bioapplications is graphene oxide (GO), a graphite derivative based on one or only a few layers of aromatic sp2 hybridized carbon atoms, which are partially functionalized with oxygen rich groups (mainly carboxylic acid, hydroxy and epoxy groups).28 As a result of the polarity of its oxidized functional groups, GO is very stable in water suspensions unlike the hydrophobic graphene, reduced GO and carbon nanotubes. This property makes GO suitable for biomedical applications such as drug delivery, biosensors, phototherapy and tissue engineering.29 For instance, nowadays it is well-known that hydrogel/GO nanocomposite hydrogels exhibit enhanced mechanical properties compared to pure hydrogels.30 When the polymers used for the hydrogels have hydrogen donor/acceptor functional groups in their structure (e.g. amido, hydroxy, amino or carboxyl groups), GO can further act as a physical crosslinker by multiple hydrogen bonds interactions.31 This characteristic could explain the superior mechanical properties of GO Page 5 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 6 nanocomposite hydrogels. Another relevant feature of GO is its bioactivity,29 motivating its incorporation into biomaterials for antimicrobial purposes. Several antimicrobial polymer/GO nanocomposites have been produced based on different bactericidal mechanisms proposed for pure GO.32 Regarding the MSC response, scaffolds based on polymer/GO nanocomposites have been mainly prepared to analyse their osteogenic effect,33 although the MSCs differentiation to other cell phenotypes has also been explored during the last years.29 Indeed, biomaterials based on GO can further enhance neurogenic,34 adipogenic35 and chondrogenic36 differentiation. The recent work by Zhou et al. is of particular interest, where a photocrosslinkable hydrogel bioink based on GEL-MA, poly (ethylene glycol) diacrylate and GO was used for 3D bioprinting scaffolds. The presence of GO in the printed MSC-loaded constructs increased the cell viability and the chondrogenic markers expression.37 These results show a promising future of GO containing hydrogels in cartilage regenerative medicine applications. The usual approach for the chondrogenic differentiation of MSCs in cartilage tissue engineering is the employment of a biochemical cocktail as chondrogenic medium. These pro-chondrogenesis agents include transforming growth factor-β (TGFβ), dexamethasone, ascorbic acid, sodium pyruvate, among other supplements.38 In particular, TGFβ is a family of cytokines which play a mediator role of signalling cascades for chondrogenic differentiation and keeping of differentiated chondrocyte phenotype.39 However, the use of TGFβ as chondrogenic supplement for hMSCs could take undesired pathways such as osteogenic and hypertrophic chondrocyte differentiation,40 tumorigenesis and metastasis in human cancer.41 These aspects are part of the issues related with MSCs therapy. Therefore, the development of regenerative therapies methods free of exogenous growth factors is necessary for safer and less expensive clinical translation of tissue engineering. Page 6 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 7 Herein, a novel nanocomposite hydrogel based on a crosslinked network of ALG, CS and GEL filled with GO is developed as ink for 3D printing scaffolds fabrication. The effect of ink composition on printing quality, cytocompatibility and the intrinsically chondroinductive effect of scaffolds was evaluated varying the GO concentration in the inks. EXPERIMENTAL SECTION Materials and reagents for hydrogel nanocomposite synthesis The biopolymers alginic acid sodium salt from brown algae (ALG), Chondroitin sulfate A sodium salt from bovine trachea (CS) and gelatin from porcine skin (GEL) BioReagent grades were purchased from Sigma-Aldrich (Norway, China and USA respectively) and used as received. 2-Aminoethyl methacrylate hydrochloride (AEMA), N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(Nmorpholino)ethanesulfonic acid (MES), methacrylic anhydride (MAA), 2-Hydroxy-4′-(2hydroxyethoxy)-2-methylpropiophenone photoinitiator (PI), graphite, potassium permanganate, sodium nitrate, calcium chloride, sulfuric acid, hydrogen peroxide, ethanol and deuterium oxide were purchased from Sigma-Aldrich or Merck .Millipore. Dialysis tube Spectra/Por® 1 (MWCO= 6-8 kDa, 50 mm flat width) utilized in purification of methacrylated biopolymers was purchased from Spectrum Laboratories. Type I (ultrapure) water was used for synthesis and purification. Spectroscopic characterization ATR-FTIR spectra were acquired from an Agilent Cary 630 FTIR spectrometer. The Raman spectroscopy study was carried out on a Horiba HR Evolution micro-spectrometer coupled to an Olympus optical microscope with a 100X objective, using a 532 nm laser, 107 mW source power and using the LabSpec6 software to acquire the spectrograms (three scans accumulated with 90 s Page 7 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 8 of acquisition). Proton NMR analysis was performed on a Bruker AVANCE III HD-400 spectrometer, experiments were measured to solutions of samples in deuterium oxide and the obtained FID data were processed with MestReNova. UV-VIS spectrum of GO was obtained using a Rayleigh UV-1601 spectrophotometer (10 mm optical path quartz cuvette). Microstructural characterization X-ray diffraction measurements were taken using Cu Kα radiation with a Bruker D8 Advance diffractometer. TEM images were acquired with a FEI Tecnai F20 S/TEM electron microscope, mounting the sample on a Holey Carbon on 300 mesh Cu grid. FE-SEM images were acquired with a FEI QuantaTM FEG SEM electron microscope. To measure the GO sheet lateral size, a silicon wafer (50 mm of diameter) was treated with plasma and spin coated with 200 µL of 50 µg mL-1 of aqueous suspension of GO at 1500 rpm during 2 min. The lateral size distribution was measured by FE-SEM image analysis of this silicon wafer coated with GO, using ImageJ software. Optical microscopy characterization Polarized light microscopy (POM) studies were performed on a Leica DMLS microscope equipped with a polarizer/analyzer system and a full-wave retardation plate. Cell culture imaging was performed with a Leica DM IL LED phase contrast/fluorescence microscope. Both instruments were equipped with a Leica MC170 HD digital camera for microphotography acquisition. Frequency distribution histogram of cells direction were plotted using the directionality plugin in Fiji/ImageJ (ImageJ software, USA).42 Chemical modification of biopolymers and synthesis of graphene oxide. The three biopolymers ALG, CS and GEL were functionalized with polymerizable methacrylate groups to obtain ALG-MA, CS-MA and GEL-MA, respectively. The photocrosslinking of the Page 8 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 15 time, the samples were washed with DPBS and observed at fluorescence microscope (each sample in triplicate). Immunofluorescence analysis The immunofluorescence staining of chondrogenic markers for collagen type II, aggrecan and SOX 9, was performed for hADMSCs (passage 9, 40000 cells/scaffold) cultured on 3D printed scaffolds after 28 days in CCM. Immunostaining was carried out as follows: scaffolds were washed twice in PBS at 37 °C, fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min and washed twice in PBS. Cells on scaffolds were permeabilized with Triton X-100 (SigmaAldrich) 0.25 % in PBS-Gly (15 g glycine/100 mL PBS, Sigma-Aldrich) for 10 min, then they were washed thrice in PBS-Gly. Scaffolds were treated with blocking buffer (6% bovine serum albumin in PBS-Gly, Sigma-Aldrich) for 45 min and incubated overnight at 4 °C in rabbit polyclonal antibody against collagen type II (ab34712, 1/100), aggrecan (ab36861, 1/100), or SOX 9 (ab3697, 1:100) (Abcam). After washing them three times in PBS-Gly, the scaffolds were then incubated in a secondary antibody (1/100, Alexa Fluor 488 goat anti-rabbit IgG H&L ab150077, Abcam) for 1 h at room temperature. Nuclei and F actin were counterstained using DAPI stain and TRITC-phalloidin (Life Technologies) respectively as is described above. Statistical analysis The measured data of water content, compression moduli and alamarBlueTM assay was expressed as arithmetic mean ± standard deviation (SD). The analysis of variance (ANOVA) and the Tukey’s post-test were performed to assess statistical significance between groups using OriginPro 8.6 (OriginLab) or Prism 6 (GraphPad). Page 15 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 16 RESULTS AND DISCUSSION Characterization of the modified biopolymers Page 16 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 17 The different biopolymers (ALG, CS and GEL) were modified with methacryloyl moieties (MA) Page 17 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 18 to obtain a high degree of functional groups able to photocrosslink, as reported previously.43 The methacrylation of biopolymers was confirmed and quantified by ATR/FTIR and 1H NMR spectroscopy. Figure 1 shows the FTIR spectra of the three biopolymers before and after methacrylation, where it is possible to observe a shoulder in the spectra of ALG-MA at 1707 cm1, and a hypsochromic shift of the carbonyl band to 1636 cm-1 in CS-MA due to the incorporation of 2-amidoethyl methacrylate groups. In addition, both polysaccharides exhibit their characteristic stretching vibration bands of O-H bonds (around 3300 cm-1), Csp3-H bonds (around 2900 cm-1), C=O bonds (around 1600 cm-1) and C-O bonds (around 1020 cm-1). The FTIR spectra of GEL and GEL-MA do not show major differences, both displaying stretching vibration bands of O-H and N-H bonds (around 3290 cm-1), Csp2-H bonds (at 3070 cm-1), Csp3-H bonds (at 2940 cm-1), C=O Figure 1. ATR/FTIR spectra of biopolymers before and after methacrylation. Page 18 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 19 bonds (around 1630 cm-1) and C=C bonds (around 1530 cm-1). The bands of methacryloyl groups in GEL-MA are likely overlapped with the intense signal of the gelatin backbone bands. The 1H NMR analysis further confirmed functionalization of the biopolymers (Figure 2). The spectra of ALG-MA and CS-MA shows the characteristic doublets of vinyl protons at 5.75 and 6.14 ppm, multiplets of methylene protons at 3.39 and 3.55 ppm and methyl protons singlet at 1.94 ppm, corresponding to amidoethyl methacrylate functionalities. The 1H NMR spectrum of GELMA shows peaks of vinyl protons of methacrylamide groups at 5.44 and 5.67 ppm, an additional doublet of vinyl protons at 5.75 and 6.14 ppm corresponding to methacrylate groups (secondary methacrylation reaction on hydroxy groups of GEL),46 and the singlet of methyl protons at 1.94 ppm. Page 19 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 20 The degree of methacrylation of biopolymers was determined from the 1H NMR spectra by the integration of the respective peaks affected by the chemical modification on the biopolymer after and before the methacrylation (for calculations details see Supporting Information). In ALG, the peak with a chemical shift of 5.04 ppm (Figure S1) corresponds to the hydrogen atoms bonded to anomeric carbons in the polysaccharide that is used as reference to integrate. The changes of integration of the peak at 4.48 and 3.78 ppm (G5 and M5 peaks in Figure S1) allows the calculation of the degree of methacrylation respective to carboxylate groups (Figure S2). These peaks correspond to the hydrogen atoms bonded to the carbon atoms with carboxylate groups in ALG. Figure 2. 1H NMR spectra of methacrylated biopolymers. Characteristic peaks of methacryloyl moieties are highlighted. Page 20 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 21 The 1H NMR spectrum of CS showed in Figure S3 displays the reference peak used for the degree of methacrylation calculation, corresponding to methyl protons of N-acetyl groups in sulfated galactosamine saccharide in CS monomer. The degree of methacrylation in CS-MA is determined by the integral ratio of the methyl protons of the methacrylate groups (1.95 ppm) to the methyl protons of the N-acetyl groups (2.06 ppm) in the CS-MA spectrum of Figure 2. The degree of methacrylation of GEL-MA (relative to free amino groups) is determined by changes in the integral of the peak at 3.02 ppm. This peak corresponds to the methylene protons bonded to the carbon atoms with free amino groups in the lysine segments of GEL. The integral of the peaks at 7.29 and 7.37 ppm, corresponding to the aromatic protons of the phenylalanine segments, were used as reference (Figure S5). Based on this methodology, the degrees of methacrylation were found to be 27 % for ALG-MA, 50 % for CS-MA and 100 % for GEL-MA, where the peak at 3.02 ppm is completely shifted and overlapped with the peak at 3.24 ppm and the peaks of free amino group protons disappear (Figure S6). These modified polymers were able to form a stable hydrogel by test tube inversion method (Figure S7), after 5 minutes of irradiation with an UV lamp using a weight ratio of ALG-MA, CS-MA and GEL-MA of 1:1:1 in presence of a photoinitiator. Characterization of graphene oxide The chemical functionalities of synthesized GO were characterized by FTIR and UV-VIS spectroscopy, whereas its microstructure was studied by XRD, TEM and POM. The FT-IR spectrum of GO (Figure 3a) displays a broadband around 3200 cm-1, corresponding to the stretching vibrational mode of O-H bonds of the hydroxyl groups. The peaks associated with the stretching of C=O bonds of carbonyl groups, double bonds C=C from aromatic carbon backbone and C-O bonds from epoxy and hydroxyl groups, are further observed at 1712 cm-1, 1618 cm-1 and 1040 cm-1, respectively. Figure 2. 1H NMR spectra of methacrylated biopolymers. Characteristic peaks of methacryloyl moieties are highlighted in squares. Page 21 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 22 The UV-Visible spectrum of a GO suspension in water shows a main peak around 230 nm Figure 3. Spectroscopic characterization of synthetized GO: (a) ATR/FTIR spectra of GO (black line) and graphite precursor (red line). (b) UV-VIS spectrum of GO measured on a water suspension 0.05 mg mL-1. (c) Raman spectra of GO and graphite precursor where the D and G bands are indicated in red and blue respectively and its ratio of intensities are shown inset. Page 22 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 23 corresponding to π→π* electronic transitions in the aromatic basal plane and a shoulder around 300 nm consistent with n→π* transitions due to oxygen atoms present in the functional groups (Figure 3b). Figure 3c displays the Raman spectra of GO and its graphite precursor, where the characteristic D band around 1357 cm-1 and G band around 1585 cm-1 were observed in the GO spectrum. The D band is attributed to defects produced after the oxidation of graphite, such as hydroxyl, epoxy and carbonyl groups. The G band corresponds to a first order scattering of the E2g mode of the graphitic structure.47 The intensity ratio between D and G peaks of GO spectrum is ~ 8-fold higher than graphite spectrum value, showing concordance with the structural modification due to the oxidation reaction. Figure 4 displays the XRD patterns of graphite and GO showing that the oxidation and exfoliation process during GO synthesis produced an increment in the interlayer distance. While graphite presents an intense characteristic peak at 26.38° (2θ, layer distance d = 0.34 nm), GO Figure 4. XRD powder diffractograms of GO (solid line) and graphite precursor (dotted line). Page 23 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 24 exhibits a peak at 12.37 ° (2θ, d= 0.71 nm) corresponding to 001 plane reflections of few-layer GO. Residual graphitic structure peaks were not observed in GO by XRD. TEM images of synthesized GO confirmed the 2D layered sheets morphology (Figure 5a) and the analysis of FESEM images allowed the determination of the lateral size of GO sheets of ~ 1 µm (Figure 5b). Based on these results, we conclude that the oxidation process of graphite was able to produce GO by adding oxygen-containing functional groups and disrupting the interlayer order. The liquid crystal (LC) property of GO in aqueous suspensions is an unexplored feature in tissue engineering, despite the high potential to template biomaterials. 48,49 Figure 5c-f shows the LC nature of our GO by means of POM, where it is possible to observe a birefringent texture in an aqueous suspension having a concentration of ~ 10 mg mL-1 of GO (Figure 5c-d), and in a wetted GO flake macroparticle (Figure 5e-f) when the samples are observed between crossed polarizer/analyser (Figure 5d and 5f). The LC order of GO sheets in aqueous suspensions allows the obtention of anisotropic 2D flakes or papers, and 1D annealing under determined conditions.50 Page 24 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 31 few layers are printed, more CaCl2 solution is added to ensure the crosslinking of ALG and ALGPage 31 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 32 MA. The 3D printed scaffolds are then irradiated with UV light for secondary photocrosslinking Page 32 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 33 and washed afterward with deionized water and freeze-dried. Figure 6a-c displays digital pictures Page 33 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 34 of the lyophilized 3D printed scaffolds using the three ink formulations. Under the same printing Page 34 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 35 conditions (optimized for the performance with ACG ink meaning a printing speed = 50 mm s-1, Page 35 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 36 extrusion pressure = 1 bar and 25 G needle tip), the scaffolds printed with inks containing GO show a noticeable higher shape fidelity and spatial resolution than ink without GO. FE-SEM images analysis show a flattening of the thread in the scaffolds having GO after the freeze-drying process (Figure 6e-f). The morphologic analysis of the surface on strands show an increment in the roughness when GO is added (Figure 6g-i). While a smooth surface is observed in scaffold with ACG composition, fibrillar and bumped micro-topography are observed on the surface of 3D printed scaffolds using ACG/GO01 and ACG/GO1 inks, respectively. Figure 6. Full-size appearance of lyophilized scaffolds 3D printed with (a) ACG, (b) ACG/GO0.1 and (c) ACG/GO1 inks. FE-SEM images of threads surfaces: (d and g) ACG, (e and h) ACG/GO0.1 and (f and i) ACG/GO1 3D printed scaffolds. Page 36 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 37 The anisotropy features in the threads of 3D printed scaffolds were studied by POM. The asprinted strands and the corresponding freeze-dried/rehydrated threads of the scaffolds without GO (ACG) did not show significant birefringence (Figure S8a-c), indicating the isotropic nature of this threads. Scaffolds with a low GO concentration (ACG/GO01) did not also show significant birefringence in the as-printed threads (Figure S8e), although a weak birefringence with grooved shape was observed on the threads after the freeze-drying/rehydration process, in accordance with the microfibril topography observed in the Figure 6h. This result is indicative of partly ordered and localized anisotropy along the ACG/GO01 threads (Figure S8f). Figure 7 shows POM images of a 3D printed thread produced using ACG/GO1 ink. Depending on the directional angle of the thread, it exhibits intense birefringence with red or blue color interference when the sample is observed with crossed polarizer/analyzer and a first order (λ) retardation plate. While the thread does not exhibit birefringence at 0 ° and 90 ° with respect to the polarizer (N-S and W-E columns in Figure 7), the thread acquires a first order red color (fast axis) when the thread orientation is Southwest-Northeast (SW-NE, Figure 7 f and j), and a second order blue (slow axis) when the thread orientation is Northwest-Southeast (NW-SE, Figure 7 h and i).The homogeneous birefringence in the thread indicates an anisotropic molecular order along the major axis. This order was observed for threads printed with ACG/GO1 in both as-printed (Figure 7a-h) and after freeze-drying/rehydration process (Figure 7i-l), and differences were not observed in the thread after (only ionocrosslinking) and before photocrosslinking (as-printed). Compounds with LC mesophases can yield this kind of anisotropic microfibers 52 or induce the alignment of polymeric chains during the fiber fabrication process.53 Aqueous GO suspensions have exhibited a stable lyotropic nematic mesophase (with a volume fraction of 100 %) in concentrations higher than 8 mg mL-1.54 Below this critical concentration, the isotropic and nematic phases co-exists, showing Page 37 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 38 only ~ 3 % of nematic mesophase at GO concentration of 1 mg mL-1. However, when a GO suspension at low concentration (for instance < 1 mg mL-1) passes through a tube at high velocity, such as the needle of a 3D printer, the GO sheets can exhibit alignment induced by the flow.55 Furthermore, recent studies show that lyotropic LC properties of GO are stabilized, and the critical concentration of mesophase formation is lowered, by the influence of alginate in composites.56 For this reason, it is likely that the micro-extrusion process and the LC mesophase of GO trigger a Figure 7. POM images of anisotropic threads of scaffolds 3D printed with ACG/GO1 ink. (a-d) POM images of an as-printed thread observed with different angles between crossed polarizer/analyzer filters, (e-h) as-printed thread observed with a retardation plate between crossed polarizer/analyzer filters and (i-l) rehydrated thread after freeze-drying process, observed with a retardation plate between crossed polarizer/analyzer filters. Scale bar: 400 μm, N-S: North-South, SW-NE: Southwest-Northeast, W-E: West-East and NW-SE: NorthwestSoutheast directions. Page 38 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 39 preferential molecular orientation along the 3D printed threads. Anisotropic structures such as fibers and films are naturally found in the body tissue, and for this reason, biomimetic anisotropic scaffolds for muscle, neural and tendon tissues have attracted attention.57 In recent years, some tissue engineering researches have been focusing their work on the development of anisotropic scaffolds by LC template to mimic the natural environment and architecture of the tissue.58 Therefore, GO appears as a novel filler triggering anisotropic behavior in biopolymer composites. Water content and swelling ratio of scaffolds The water content of photocrosslinked scaffolds and water uptake capacity after lyophilisation was studied for three compositions (ACG, ACG/GO01 and ACG/GO1). The Figure 8a displays the water mass fraction of as-photocrosslinked and freeze-dried/rehydrated scaffolds. The asphotocrosslinked scaffolds exhibit high water mass fraction (~ 0.96) and no differences between the three samples was observed. When the scaffolds are rehydrated after lyophilisation, the water Figure 8. Water content in the scaffolds: (a) Water mass fracction in scaffolds after photocrosslinking (blue bars) and after freeze-driying/rehydratation process (red bars). (b) Swelling ratio of scaffolds. ns: not significant, *: p<0.05, ***: p<0.001 and ****: p<0.0001. Page 39 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 40 content in scaffolds decrease between 6-10 % and the water absorption capacity in nanocomposite scaffolds is significantly superior than ACG scaffolds. Furthermore, the swelling ratio of GO containing scaffolds are between 50-66 % higher than ACG scaffolds, being able to increase their mass up to 10-fold by absorbing water (Figure 8b). These results suggest that the incorporation of GO into the biopolymeric network increases the hydrophilicity and water retention, probably due to the presence of polar oxygen rich functional groups that could interact with water molecules by hydrogen bonding. Rheological properties of inks The rheological behaviour of ink formulations used for 3D printing scaffold fabrication was studied at 4 °C. The pure hydrogel matrix and the composites presented a similar viscosity with a shear thinning behaviour during the whole range of applied shear rates (Figure 9a). Despite the low concentration of GO used, the nanocomposites displayed lower viscosities than the pure hydrogel, which is likely associated with the reduction of the polymer free volume59 or the adsorption of polymer molecules on the particle surface.60 Figure 9b shows the storage and the loss moduli as a function of frequency for the three ink formulations. All inks exhibit liquid-like hydrogel behaviours before crosslinking with low modulus values (between 10 and 100 Pa) and tan δ = G’’/G’ > 1. The data of the storage and the loss moduli fit with a Maxwell scaling law, this means that at low frequencies (ω → 0), G’ ~ ω2 with a slope = 1 and G’’ ~ ω with a slope = 2. After the addition of a calcium chloride solution (t= 60 s), a clear transition to solid-like characteristics is observed with a drastic increase in the moduli of several orders of magnitude, while G’ crosses G’’ (tan δ < 1), as displayed in the kinetic curves of ionotropic crosslinking (Figure 9d). Figure 9c shows the thixotropy study made in order to mimic the shear rate effect on the viscosity of inks during the 3D printing process.61 The first 60 s at a low shear rate of 0.1 s-1 Page 40 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 47 fluorescence staining assay. For alamarBlueTM assay, hADMSCs (passage 9) were seeded on the Page 47 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 48 3D printed scaffolds, utilizing pure ALG scaffold as control. Figure 11a shows the evolution of Page 48 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 49 the hADMSCs proliferation (displayed as mean fluorescence intensity) on 3D scaffolds during 14 Page 49 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 50 days. The average cell viability in each scaffold is not significantly different from controls (at the Page 50 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 51 0.05 level) during the first three days, suggesting that the chemical composition of the different Page 51 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 52 inks is cytocompatible with hADMSCs at early stage, independent of the bioconjugation and the Page 52 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 53 presence of GO. However, after one week the proliferation of hADMSCs on bioconjugated Page 53 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 54 scaffolds was significantly superior as compared with pure ALG control scaffolds, as the cell Page 54 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 55 amount decreases considerably on the latter. Noteworthy, ACG/GO0.1 scaffolds presented higher Page 55 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 56 cell proliferation than ACG and ACG/GO1 scaffolds. The low cell viability in ALG scaffolds can be explained by cells aggregation as shown in Figure 11b, meaning that in these scaffolds the cellcell interaction is preferred rather cell-material interaction.63 These cellular aggregates can be detached from the scaffolds during both, the successive culture media changes through aspiration and the washing procedure of the assay, which causes a dramatic decrease on the cells number. The cell proliferation on the scaffolds increases significantly, when ALG is bioconjugate with CS and GEL. This increment in proliferation means that cells prefer to attach on the scaffold surface rather than to other cells. Noteworthy, in the ACG/GO0.1 scaffolds the cells proliferate homogeneously on the surface and around the threads of the scaffold without the formation of cell aggregates (Figure 11d). By adding more GO, the high proliferation produced not only cells distributed on the scaffold surface but also small cell aggregates (Figure 11e). After 14 days, the Figure 11. Proliferation of hADMSCs on 3D printed scaffolds. (a) Cell proliferation quantification by alamarBlueTM assay (*: p<0.05, **: p<0.01 and ***: p<0.001). Contrast phase microphotography of cell-seeded scaffolds at day 7: (b) ALG, (c) ACG, (d) ACG/GO0.1 and (e) ACG/GO1. Cell aggregates are denoted with arrows (scale bar: 100 μm). Page 56 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 63 In a recent study, Shen et al. evaluated the effect of GO nanocomposite hydrogels in exogenous TGFβ–free chondrogenesis of hMSCs.36 This pioneer work evidenced the chondroinductive property of GO and suggests that GO is able to concentrate locally pro-chondrogenic biomolecules in the cellular environment. A chondrogenic culture medium supplemented with prochondrogenesis agents (insulin-transferrin-selenium (ITS), dexamethasone, L-ascorbic acid 2phosphate, L-proline) was used in that study, however, our study shows the intrinsic chondroinductive effect of the developed biomaterials without the need of any exogenous prochondrogenic factor in the culture medium. The secretion of collagen type II after 28 days in the ACG/GO0.1 scaffolds show high similarity in density and anisotropic distribution (detailed immunofluorescence image in Figure S11) with the observed in immunofluorescent analysis of human cartilage tissue and tissue engineered neocartilage.70 The results suggest that ACG/GO0.1 scaffolds have the optimal properties for printability, biocompatibility, cell proliferation and interconnected neocartilage ECM deposition, where the biopolymer matrix and GO acts synergistically in the hADMSCs chondrogenic differentiation. Page 63 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 64 Figure 15. Fluorescence images of immunostaining for chondrogenic markers (green) collagen type II (COLL II), aggrecan (ACAN) and SOX 9 for the 3D printed scaffolds: ACG (top), ACG/GO0.1 (middle) and ACG/GO1 (bottom), after 28 days of culture. Cytoskeleton F actin and nuclei counter staining are showed in red and blue respectively (Scale bar: 500 μm). Page 64 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 65 CONCLUSIONS In this work we present novel bioconjugated nanocomposite hydrogels based on alginate, crosslinked with gelatin and chondroitin sulfate, and GO particles as inks for 3D printing scaffolds. The incorporation of GO into the ink formulation enhanced its printability, obtaining scaffolds with higher shape fidelity and resolution than ink without GO, due to an enhanced thixotropic behaviour. Threads printed with ACG/GO1 result in anisotropic fibers, probably induced by liquid crystalline properties of GO, with interesting projections for tissue engineering where cellular alignment is required. 3D printed bioconjugated scaffolds showed to be cytocompatible with hADMSCs and samples with GO presented outstanding cell proliferation, alignment and distribution in the scaffolds. Immunostaining analysis indicated that the bioconjugate alginate polymer matrix is intrinsically chondroinductive on hADMSCs differentiation after four weeks culture in non-chondrogenic medium. The superior biocompatibility and bioactivity of the 3D printed scaffolds based on our bioconjugated nanocomposite with hADMSCs, position them as candidates for cartilage tissue engineering. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/xxxxxx. 1H NMR characterization of used pure commercial biopolymers and degree of methacrylation determination for ALG-MA, CS-MA and GEL-MA. Photocrosslinking test. Cumulative GO release. Negative controls images for anisotropy and immunofluorescence studies. Stereomicroscopy images of scaffolds after 35 days. Immunofluorescence image of anisotropic Page 65 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 66 cell organization of neocartilage in ACG/GO01 scaffold (PDF). Video showing the 3D printing process (WMV). AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (F.O.-M); [email protected] (H.P.); tel.: +56229780795 ORCID Felipe Olate-Moya: 0000-0002-2000-0708. Humberto Palza: 0000-0001-5246-6791. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) under FONDECYT Project 1150130, FONDEQUIP Projects EQM150101, EQM170103 and EQM140012, CONICYT Beca de Doctorado Nacional grant 21150039 and Millennium Science Initiative of the Ministry of Economy, Development and Tourism, grant “Nuclei for Soft Smart Mechanical Metamaterials” ACKNOWLEDGMENT The authors thank the financial support of CONICYT under FONDECYT Project 1150130, FONDEQUIP Projects EQM150101, EQM170103 and EQM140012, and funding from Page 66 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 67 Millennium Science Initiative of the Ministry of Economy, Development and Tourism, grant “Nuclei for Soft Smart Mechanical Metamaterials”. F. O.-M. thanks to CONICYT Beca de Doctorado Nacional 21150039. ABBREVIATIONS AEMA, 2-Aminoethyl methacrylate hydrochloride; ALG, alginate; ALG-MA, alginate methacrylated; ATR-FTIR, attenuated total reflection Fourier transform infrared spectroscopy; CCM, conditioned culture medium; CS, chondroitin sulfate; CS-MA, chondroitin sulfate methacrylated; DAPI, 4′, 6-diamidino-2-phenylindole; ECM, extracellular matrix; EDC, N-(3dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; FBS, fetal bovine serum; FE-SEM, field emission scanning electron microscopy; FITC, fluorescein isotiocyanate; GAG, glycosaminoglycan; GEL, gelatin; GEL-MA, methacryloyl gelatin; Gly, glycine; GO, graphene oxide; hADMSC, human adipose tissue derived mesenchymal stem cell, hMSC, human mesenchymal stem cell; 1H NMR, proton nuclear magnetic resonance; LVR, linear viscoelastic region; MAA, methacrylic anhydride; MEMα, minimum essential medium alpha; MWCO, molecular weight cut-off; MES, 2-(N-morpholino)ethanesulfonic acid; NHS, Nhydroxysuccinimide; PI, 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone photoinitiator; PBS, phosphate buffered saline; PG, proteoglycan; POM, polarized light microscopy;RGD, arginylglycylaspartic acid; STL, standard triangle language; TEM, transmission electron microscopy; TRITC, tetramethylrhodamine; UV-VIS, ultraviolet–visible spectroscopy; XRD, X-ray diffraction. 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Page 79 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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 80 For Table of Contents Only Page 80 of 80 ACS Paragon Plus Environment ACS Applied Materials & Interfaces 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