Graphene OxideâA Tool for the Preparation of Chemically

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Biological and Medical Applications of Materials and Interfaces

Graphene Oxide–A Tool for Preparation of Chemically Crosslinking Free Alginate-Chitosan-Collagen Scaffold for Bone Tissue Engineering Elayaraja Kolanthai, Abinaya Sindu P, Deepak Kumar Khajuria, Sarath Chandra Veerla, Dhandapani Kuppuswamy, Luiz Henrique Catalani, and D. Roy Mahapatra ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00699 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

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ACS Applied Materials & Interfaces

Graphene Oxide–A Tool for Preparation of Chemically Crosslinking Free Alginate-Chitosan-Collagen Scaffold for Bone Tissue Engineering

Elayaraja Kolanthai,a,b,* P. Abinaya Sindu,c Deepak Kumar Khajuria,b Sarath Chandra Veerla,b Dhandapani Kuppuswamy,d Luiz Henrique Catalani,b D. Roy Mahapatraa,*

a

Laboratory for Integrative Multiscale Engineering Materials and Systems, Department of

Aerospace Engineering, Indian Institute of Science, Bangalore 560012, India b

Departamento de Química Fundamental, Instituto de Química, Universityof São Paulo, Av.

Prof. LineuPrestes, 784, 05508-000 São Paulo, Brazil c

Centre for Biotechnology, Anna University, Chennai 600 025, India

d

Cardiology Division, Department of Medicine, Gazes Cardiac Research Institute, Medical

University of South Carolina, Charleston, SC 29425-2221, USA.

Key words: Graphene oxide, Alginate, Collagen, Chitosan, Hydrogel, Bone tissue engineering *Corresponding author´s Tel. No: +91-80-22932419 E-mail id: [email protected] E-mail id: [email protected]

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Abstract Developing a biodegradable scaffold remains a major challenge in bone tissue engineering. This study was aimed at developing novel alginate-chitosan-collagen (SA-CS-Col) based composite scaffolds consisting of graphene oxide (GO) to enrich porous structures, elicited by freeze-drying technique. To characterize porosity, water absorption and compressive modulus, GO scaffolds (SA-CS-Col-GO) were prepared with and without Ca2+-mediated crosslinking (chemical crosslinking) and analyzed using Raman, FTIR, XRD, and SEM techniques. Incorporation of GO into SA-CS-Col matrix increased both crosslinking density as indicated by the reduction of crystalline peaks in the XRD patterns and polyelectrolyte ion complex as confirmed by the FTIR. Graphene oxide scaffolds showed increased mechanical properties which were further increased for chemically crosslinked scaffolds. All scaffolds bared interconnected pores of 10 µm to 250 µm range. By increasing the crosslinking density with Ca2+, a decrease in porosity/swelling ratio was observed. Moreover, SA-CS-Col-GO scaffold with or without chemical crosslinking was more stable as compared to SA-CS or SA-CS-Col scaffolds when placed in aqueous solution. To perform in vitro biochemical studies, mouse osteoblast cells were grown on various scaffolds and evaluated for cell proliferation by using MTT assay, and mineralization and differentiation by alizarin red S staining. These measurements showed a significant increase for cells attached to the SA-CS-Col-GO scaffold, compared to SA-CS or SA-CS-Col composites. However, chemical crosslinking of SA-CS-ColGO showed no effect on the osteogenic ability of osteoblasts. These studies indicate the potential use of GO to prepare free SA-CS-Col scaffolds with preserved porous structure with elongated collagen fibrils, and that these composites, which are biocompatible and stable in a biological medium, could be used for application in engineering bone tissue.


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1. Introduction Engineering tissues is a rapidly growing field where biological substitutes are used for tissue and organ regeneration in human body. Tissue regeneration requires biodegradable scaffold with satisfactory mechanical strength to support cell attachment, differentiation, and growth with passable biocompatibility1-3. In bone tissue engineering, biodegradable scaffolds with suitable mechanical properties act as a temporary template for the native extracellular matrix until the osteoblast cells produce their own bone matrix4. Different scaffolds are employed in engineering bone tissue applications like ceramic, hydrogel-based scaffold and biodegradable polymer5. Among them, hydrogel-based scaffolds have several advantages due to their biocompatibility, flexibility and hydrophilic surface nature which provides better cell attachment, growth, and differentiation6. Sodium alginate (SA) obtained from brown sea algae is a long chain polysaccharide made up of guluronate and mannuronate saccharide units. SA is known to form a stable gel when divalent cations like calcium are added to an aqueous solution of SA at room temperature. This kind of polysaccharide is frequently used as biomedical implants due to the presence of Dguluronic acid as the major sugar unit in their structure7-10. However, pure SA has limited applications in engineering bone tissue due to limited mechanical properties and cellular interactions11. To overcome these limitations, several SA based composites like SA–chitosan12, SA–collagen13, SA–gelatin14, and SA–hydroxyapatite11 were developed for engineering bone tissue application. Chitosan (CS) is a polysaccharide of cationic nature and it is primarily composed of (1,4) 2-amine 2-deoxy D-glucopyranose and a low level of N-acetyl-d-glucosamine. Chitosan is a deacetylated derivate of chitin and has cell adhesive properties. Chitosan is broadly used for



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engineering bone tissue owing to its accelerating wound healing nature, antimicrobial and bone regeneration activity15-16. The positively charged CS endows electrostatic interactions in the presence of negatively charged molecules. For example, electrostatic interactions between protonated amine group of CS and carboxylate moieties of SA results in the formation of a polyelectrolyte complex that could be used in the form of membrane, capsule, fiber or scaffold9, 17-19

. Collagen (Col) is widely accepted materials in bone tissue engineering applications20.

Collagen contains distinct adhesion sites bearing arginine-glycine-aspartic acid (RGD) rich regions that promote cell adhesion and enhance cell proliferation and differentiation21. However, as a scaffold, Col presents limitations owing to its low mechanical strength with swift biodegradation22. Blending of Col with biodegradable polymers is known to produce scaffolds with compelling mechanical property for applications in bone tissue engineering23. Previous studies report on the use of SA-CS-Col composites to form biodegradable scaffolds24-25. Graphene and graphene oxide (GO) are emerging carbon materials for applications in bone tissue engineering26. Also, it is used to deliver antibiotics and anticancer drugs precisely to the affected tissues in the human body27-29. A recent study shows that the electrospun scaffold of GO/gelatin/poly(lactic-co-glycolic acid) composite accelerates murine MC3T3 cell growth and differentiation5. The incorporation of GO in poly(3-hydroxybutyrate-co-4-hydroxybutyrate) polymer reduces the fiber diameter in electrospun fiber mat but it increases mechanical strength and hydrophilicity of the scaffold. Also, this scaffold shows

improvement of cellular

performance and osteogenic differentiation in in vivo bone repair studies tested in a rat model.30Also, several studies demonstrate that GO can improve cellular behavior due to enhanced mechanical properties and large surface area of polymer matrix


5,27-29, 31-32

. Study by

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Liu et al33showed that graphene coated glass slides promotes stem cell growth, attachment and osteogenic activity. Further, the GO-based poly(ethylene glycol) methyl ether-ε-caprolactoneacryloyl chloride/methacrylate chondroitin sulfate composite scaffold was used for cartilage tissue regeneration34. Graphene oxide contains several functional groups including an epoxy, carboxyl, and hydroxyl groups over its surface which help GO particles to disperse in solvents and serve as crosslinking junction for polymer-polymer and polymer-biomolecules bonds. Since GO structure features a delocalized electron arrangement, GO-based materials are endowed with fluorescence quenching ability and are also capable of absorbing near infrared radiation35-37. Further, it is noteworthy to mention that GO can be readily functionalized with several materials through their hydrophilic, p–p stacking, and electrostatic interactions5. Therefore, GO could be utilized as a new bio-bridgeing unit for biomedical implants like SA-CS-Col with enhanced biological and mechanical properties in tissue engineering. However, GO-based SA-CS-Col scaffolds have not been reported previously for engineering bone tissue applications. This study evaluated the mechanical properties, biodegradation and cytocompatibility of SA-CS-Col scaffold crosslinked or not by the addition of GO nanoparticles. First, we fabricated three different combinations of scaffolds such as SA-CS, SA-CS-Col and SA-CS-Col-GO (a novel fabricated scaffold) which could be used for engineering bone tissue. To achieve this goal, we examined the structure, morphology, mechanical properties and degradation rate of SA-CSCol-GO scaffold by Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), compressive test, porosity, and water absorption. Second, the novel SA-CS-Col-GO scaffolds were then used in mouse osteoblast cells to characterize the effect of GO-crosslinked scaffolds on cell attachment, growth and differentiation.



2.

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Materials and Methods

2.1. Materials Low molecular weight chitosan (85% deacetylated chitin with poly(D-glucosamine), sodium alginate ( 2-12 cP), collagen from bovine Achilles tendon, ascorbic acid, and β-glycerophosphate were obtained from Sigma-Aldrich (St Louis, MO, USA). Potassium permanganate, sulfuric acid (98.9 %), phosphoric acid (99%), hydrogen peroxide (99.9%), hydrogen chloride (99 %), acetic acid(99.9%) and other solvents were purchased from Merck (Kenilworth, NJ, USA). Graphite flakes were procured from Superior Company (Chicago, IL, USA). Eagle’s minimum essential medium (α-MEM), fetal bovine serum (FBS), streptomycin, penicillin, and 3(4,5dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT assay kit) were obtained

from Invitrogen

(Carlsbad, CA, USA). Mouse pre-osteoblast (MC3T3-E1 subclone-4, CRL-2593™) cells isolated from mouse calvaria were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). 2.2. Preparation of GO Modified Hummer´s method was used to prepare GO as reported previously38. In brief, the oxidization of graphite flakes was performed by mixing graphite (1g) with sulfuric acid (55 ml), orthophosphoric acid (7 ml) and potassium permanganate (6 g) and the contents were stirred continuously with a magnetic stirrer (Figure 1). The temperature was maintained at 4 ºC to manage the exothermic reaction during the addition of KMnO4. The oxidation was stopped by the addition of H2O2 solution (50 ml).The solution of highly oxidized graphites showed a bright yellow color and the oxidized graphite particles were collected after centrifuging at 5000 rpm for 1h. The particles were sequentially washed alternatively with 1 M HCl solution and sterile water for five times. These particles were washed finally with water to evade the acid from GO


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particles. Finally, GO particles were dried under vacuum for three days and then incubated at 40 ºC in a hot air oven for one day. 2.3. Preparation of SA-CS, SA-CS-Col, and SA-CS-Col-GO scaffolds Initially, the stock solutions of 2% (w/v) SA (300 ml), and 1% (wt) GO (100 ml) were prepared using deionized water and 2% (w/v) CS (300 ml) and 2% (w/v) Col (200 ml) were prepared using 0.5 M of acetic acid. Three different combinations of hydrogel scaffolds were prepared in this study. (1) SA-CS hydrogel preparation: 100 ml of CS was dropwise added to 100 ml of SA solution for 1 h under continuous stirring. (2) SA-CS-Col hydrogel preparation: 100 ml of Col was dropwise added to 100 ml of CS solution for 1 h with continuous stirring. Subsequently, 100 ml of SA solution was dropwise added to the mixture of CS-Col solution for 1 h under vigorous stirring using magnetic stirrer. (3) SA-CS-Col-GO hydrogel preparation: 1% (wt) of GO particles was uniformly dispersed in 100 ml of deionized water using probe sonicator. GO was slowly mixed with 100 ml of SA solution. The mixture of GO-SA was added dropwise into 200 ml of the CS-Col solution under vigorous stirring. SA-CS, SA-CS-Col and SA-CS-Col-GO solutions were prepared at room temperature. After preparation, these solutions were stirred continuously at 27 ºC for one day and then poured into polystyrene plates and kept at -20ºC. The frozen samples were subsequently freeze-dried at -80 ºC for one day. A schematic diagram for the preparation of the porous scaffold is shown in Figure 1. For chemical crosslinking, all freeze-dried scaffolds were soaked in ethanol for one day followed by soaking in 2% (w/v) of calcium chloride solution for one day. Finally, by using deionized water the scaffolds were washed and freeze dried for one day. The scaffolds SA-CS,



SA-CS-Col and SA-CS-Col-GO which were chemical crosslinked are referred to as c-SA-CS, cSA-CS-Col and c-SA-CS-Col-GO, respectively. The photographic images of non-crosslinked and chemically crosslinked scaffolds are shown in Figure 2. 2.4 Characterization We characterized the functional groups present in both GO and fabricated scaffolds by FTIR spectroscopy. The spectrum was recorded in ATR (attenuated total reflectance) technique of wavelength between 550 to 4000 cm-1 (64 scans for each sample) using Perkin Elmer Frontier IR/NIR spectrometer with 2 cm-1 resolution. Raman spectrum of synthesized GO particles was recorded in HORIBA LabRAM HR system using laser source with wavelength of 514 nm at 50 mW excitation power.. The XRD pattern of GO particles and scaffolds were recorded in a PANanalytical XpertPro powder diffractometer using 40 kV and 30 mA power with a CuKα source of wavelength of 1.5406 Ǻ. The scanning range of 2θ was from 5° to 60° at 1°/min scanning rate with 0.02 °/s increment step rate. The surface microscopic structures of the SACS, SA-CS-Col and novel SA-CS-Col-GO scaffolds were examined using an ESEM Quanta 200-FEI scanning electron microscopy. Further, the zeta potential and average particle size of GO particles were measured by a Malvern Zetasizer Nano ZS90 DLS (dynamic light scattering) system. The porosity of non-crosslinked and chemically crosslinked scaffolds after freezedrying was measured using liquid displacement procedure39. Round samples of diameter 10 mm and thickness 5 mm had their volume (VI) and weight (WI) determined, followed by immersion in 10 ml of ethanol. The final weight (WF) of saturated scaffolds was measured. The scaffold porosity was measured by using this equation: Porosity (%) =

(WF − WI ) (1) ρVI

where ρ= 0.789 g/cm3 denotes the density of ethanol. 8 ACS Paragon Plus Environment

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2.4.1 In vitro biodegradation The non-crosslinked and crosslinked scaffold (75 mg) were taken in 12 well culture plate and then 6 ml of lysozyme containing PBS (enzyme activity 10,000U/L) was added. These plates were incubated at 37 ºC under constant shaking (100 rpm) for two weeks. The scaffolds were gently removed from lysozyme containing PBS solution at two independent time-points and then dried at 50 ºC in hot-air oven to attain a constant weight. The percentage weight loss (WLoss) of non-crosslinked and chemically crosslinked scaffolds was measured by using the following equation: (%) WLoss = ((W0 - Wt)/W0)*100 (2) where W0 and Wt ,are the weight of scaffold before and after degradation, respectively. 2.4.2 Swelling study The swelling capacity of freeze-dried scaffolds was analyzed by immersing the samples in PBS and sterile water for one day at room temperature for complete rehydration. At pre-determined time intervals, the scaffolds were removed from the sterile water and PBS solution, and the excess solution on the surface of the scaffolds was gently dried. The weight of the scaffolds in swollen (Wss) and dried (Wds) states were measured using Sartorius weighing balance. The swelling ratio (%) of the scaffolds were calculated using the following equation =

× 100

(3)

2.4.3 Mechanical Testing The mechanical behavior of lyophilized scaffolds was analyzed in the compressive mode by a MetraviB dynamic mechanical analyzer (MaterviB DMA 100). Briefly, we used circular disc-shaped scaffolds of 10 mm diameter and 5mm thickness for mechanical testing. The scaffolds were compressed to 60% of their original thickness with a compressive strain rate of 9 ACS Paragon Plus Environment


0.01 mm/s. The compressive modulus for the stain at 10%, 20% and 40% was computed for the lyophilized scaffolds from the stress-strain curve. 2.4.4 Cell culture and viability In this study, the mouse pre-osteoblast cells were maintained at 37 °C in the culture medium α-MEM enhanced with FBS (10%), streptomycin (10 µg/ml) and penicillin (10 µg/ml) under 5% CO2 humidified atmosphere. After allowing three passages, cells with 80% confluency were trypsinized using 0.25% trypsin-EDTA and subsequently suspended in the cell culture medium containing ascorbic acid (50 µM) and β-glycerophosphate (10 mM) for osteogenic induction.40 Scaffolds in circular discs (10 mm x 3 mm diameter and thickness) were sterilized using ethanol, washed with 3x PBS solution and kept in 48 well tissue cell culture polystyrene (TCPS) plates before seeding 1x104cells/well. Mouse osteoblast grown on cell culture polystyrene surface in the absence of scaffolds served as a control. Cell proliferation on non-crosslinked and crosslinked scaffolds was evaluated at three different culture points 1, 3 and 7 days by MTT assay5.The scaffolds were stained by ethidium homodimer and calcein AM for differentiating the dead and live cells on the scaffolds surface at each culture time point. The stained cells were viewed under an Axio Imager 2Carl Zeiss fluorescent microscope. In vitro mineralization on both with and without crosslinked scaffolds and TCPS surface was determined via staining the mouse osteoblast cells using ARS (alizarin red S) on day 14. Briefly, 1x104 cells were seeded into cell culture plate containing the scaffolds and, at specified time points, the cells on the scaffold were chemically fixed with 4% PFA (paraformaldehyde). The cell/scaffold samples were stained by 2% ARS solution for 20 min. After removing ARS stain, the scaffolds were carefully washed thrice using sterile water, Scaffolds were treated with 500 µl acidic solution (sodium dodecyl sulfate 5% in 0.5 N HCl) for


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30 min to release the scaffold-bound stain. The absorbance value of the released ARS stain was measured in an ELISA (TecanInfinite® 200 PRO, USA) reader at 415 nm. 2.5 Statistical analysis All quantitative data were obtained in sextuplicates on each scaffold for all the assays. Statistical analyses for multiple comparisons of the raw data collected were obtained by one-way ANOVA (Tukey’s test) and if p < 0.05 the difference achieved is considered to be statistically significant. The results are shown as mean ± standard deviation. 3.

Results and Discussion

3.1 GO particle characterization The XRD pattern of graphite flakes showed the diffraction peak at 2θ value of 26.2º with 3.4 Å interplanar distance. The characteristic peak of graphite disappeared and a new peak appeared after oxidation, which is the characteristic (001) peak of GO at 2θ value of 10.2º with an interplanar distance of 8.6 Å (Figure. 3a). This observation could be due to the interaction of the oxygen group presence in the layers and the attached functional groups to the graphene layer. The IR spectrum of GO (Figure 3b) showed a broad peak in the region between 3700 cm-1and 2800 cm-1 owing to the stretching vibration of OH and CH2.The absorption peaks at 1732 cm-1 and 1628 cm-1 were ascribed to the C=O stretching vibration of carboxyl group and C=C stretching of sp2 hybridized crystal structure of graphite, respectively. The characteristics peaks at 1059 cm-1, 1225 cm-1 and 1369 cm-1, representing the oxygen bonded functional groups such as epoxy, alkoxy, carbonyl, and carboxyl groups, were observed in the IR spectrum of GO particles. GO displayed G and D peaks at 1594 cm-1 and 1349 cm-1 in Raman spectrum because of the 1st order E2g mode41. High intensity of D-band was due to the structural defects produced by the addition of –COOH, OH, epoxy and alkoxy functional groups in edges of graphene layer



during the oxidation process. The low-intensity vibration broad peak at 2657 cm-1 is the results of the over tone of 2D, and 2G bands with respect to the D and G bands (Figure 3c).The layer structure of GO was observed in the image obtained by SEM (Figure 3d).The particles size of GO was calculated from SEM image (S. Figure 1) which varies from 100 to 1300 nm observed in SEM image (S. Figure 1).Further, the zeta potential and the average particle size of GO were 47 mV and 784 nm, respectively(S. Figure 1). 3.2 Structural analysis of the scaffolds FTIR spectra of the pure form of SA, CS, Col and newly synthesized scaffolds are shown in the Figure4a-i. In the IR spectrum of the SA powder, the major absorption peaks at 3246 cm-1 (OH), 2922 cm-1(CH2), 1598 cm-1(COO-asymmetric), 1406 cm-1 (COO-symmetric), 1294 cm-1 (CCH and OCH), 1083 cm-1 (OCO ring), 1024 cm-1 (C-O stretching bond), 883 cm-1 (deformation of C1-H bond), and 815 cm-1(Mannuronic acid residues) confirms the presence of characteristic vibrational groups of SA 42 (Figure 4a). In the case of CS powder IR spectrum, the unique vibrational peaks specific for amide (I and II) and amino groups of CS appeared at 1651 cm-1, 1590 cm-1 and 1173 cm-1 9. These amide peaks of CS confirm the partial N deacetylation in chitin (CS) because deacetylated CS was used for this scaffold preparation. The absence of narrow absorption band at 3501 cm-1 in IR spectrum of CS showed lack of free hydroxyl groups. The characteristics vibrational peaks of polysaccharide structure in CS are observed at 1378 cm-1 (CH2 bending), 1151 cm-1 (asymmetric C-O-C stretching band), 1064 cm-1 and 1030 cm-1 (COO stretching vibration) (Figure 4b)43,44.The absorption peaks found at 3350 cm-1, 2925 cm-1, 1643 cm-1, 1550 cm-1, and 1238 cm-1 present in IR spectrum of Col are assigned precisely to the groups of amide (A, B) and amide ( I, II and III)45. The wide broad peak observed at 3450 cm-1 and the narrow sharp peak at 1450 cm-1 in Col are due to the stretching vibration of OH , and


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pyrrolidinerings46. A free N-H stretching vibrational peak of 3400 cm-1 is slightly shifted towards lower wavenumber (3350 cm-1, amide A) in Col owing to the formation of hydrogen bond, which stabilizes its helix structure47.The amide B peak of the Col (2925 cm-1) is attributed to the asymmetrical vibrational stretching mode of CH2. The amide I, II and III peaks of Col are observed at 1643 cm-1, 1549 cm-1, and 1238 cm-1. They represent primarily (i) stretching of the – C=O group of the polypeptide backbone, (ii) bending vibrations of N-H and (iii) wagging of CH2 and stretching of C-N vibration groups in glycine backbone as well as proline side chains, respectively

48

(Figure 4c). These results validate the presence of triple-helical structure in Col

used49. In IR spectrum of SA-CS scaffold, the amide II peaks intensity has increased, and the peak corresponds to amide I shifted to 1655 cm-1 from 1644 cm-1 which indicates the complete loss of amino group (Figure 4d). These variations denote the formation of a polyelectrolyte complex (SA-CS) through ionic interaction among the positively charged protonated group of NH3+ in CS and the negatively charged group of -COOH in SA. The interactions between -C=O bonds of CS and protonated –COOH groups of SA produced intermolecular hydrogen bonds in the scaffold (Figure 5a). Similarly, in the SA-CS-Col and SA-CS-Col-GO scaffolds, the bond formation was via ionic interaction (Figure 4e and f). The –OH, -NH2 and –C=O groups in Col and –OH, -COOH and –C=O groups over the GO surface can form hydrogen bonding with –NH2 and –OH in CS and –OH and –COOH in SA. The sodium alginate-chitosan scaffold crosslinked with CaCl2 showed a significant change in IR spectrum where defined peaks found at 1643 cm-1 (amide I) and 1574 cm-1 (amide II) were modified and a new peak appeared at 1621 cm-1. This might be due to the formation of network matrix by the carboxylic groups in the alginate (Figure 4g). Further, in the IR spectrum of



crosslinked SA-CS-Col, there was no change observed in the amide bands as compared with crosslinked SA-CS IR spectrum. This result helps to conclude that there is no significant effect on the triple-helical Col structure after the formation of the scaffold (Figure 4h).The addition of GO enhanced the intensity of – OH group (3200 cm-1 to 3700 cm-1) with broader band representing the intermolecular hydrogen bonding in the scaffold (Figure 4i). Its intermolecular hydrogen bonding induces excellent interfacial adhesion and improves mechanical properties50 (Figure 5b). The XRD analyses of non-crosslinked and crosslinked scaffolds are shown in Figure 6. The blend of SA-CS scaffold showed the broad crystalline 2θ peaks at 14º and 21.2º. Sodium alginate-chitosan-collagen and sodium alginate-chitosan-collagen-graphene oxide scaffolds showed a reduction in peaks intensity compared with SA-CS scaffold. This decrease might be owing to the deformation of the hydrogen bond between –NH2 and OH groups in CS on incorporation with SA, Col and GO

51

. The crystalline XRD peaks were drastically reduced

further in the scaffolds crosslinked with Ca2+ ions which indicate the increase of the amorphous phase of scaffold. Crosslinking with calcium ion has been shown to hinder hydrogen bonding between functional groups of -OH and –NH2 in CS crystal structure52. From these results, we concluded that the increase in crosslinking density between calcium ions and polymeric composites reduced the crystallinity nature of the scaffolds. 3.3 Compressive Test Figure 7 shows the stress-strain curves generated from the compressive test conducted on the non-crosslinked as well as chemically crosslinked scaffolds. The compressive modulus of the crosslinked, as well as non-crosslinked scaffolds, showed a steady growth when the strain rate increased (Table 1). These differences in the compressive modulus can be attributed to the


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diversity in the pore size (microstructure), variation in the composition of polymers including their concentration, molecular weight and density in addition to their stiffness on the fabrication of scaffolds. Compressive modulus value of SA-CS scaffold was 0.11±0.06 MPa at 10% strain with the gradual increase in the compressive strength at 20% and 40% strain rate. On incorporation of Col and GO, the mechanical property of scaffold showed greater stiffness and compressive strength with high modulus value of the resultant 3D matrices. Chitosan acts as a plasticizer with Col and the stronger intermolecular electrostatic interaction between Col and CS enhances the mechanical property of the scaffold with modulus value of 0.66±0.20 MPa at 40% strain 53. Further, reinforcement of GO increased the compressive modulus to 0.87±0.05 MPa at 40% strain due to the formation of hydrogen bond among OH and -COOH functional groups on the GO surface and with hydroxyl group of SA, and arranged a brick-and-mortar-like structure with more significant influence on the modulus value of the scaffold50. At 40% strain value, the modulus values for c-SA-CS, c-SA-CS-Col and c-SA-CS-Col-GO scaffolds were 0.81±0.01 MPa, 2.52±1.13 MPa, and5.33 ± 1.54 MPa, respectively. These results confirmed that the compressive modulus increased for chemically crosslinked scaffolds when compared to noncrosslinked counterparts.

High ionic-crosslinking density between Ca2+ ions and polymer

molecules is expected to produce the egg-box like structure with G residues of G-blocks

54

to

form a rigid composite scaffolds. The modulus value of these composites strongly matches the modulus values of human tissues, including soft collagenous, cervical spinal components, and ligaments bone. Young’s modulus values for ligamentum flavum, interspinous ligaments and elastin ligaments from bovine were1.5, 1.5 and 1.1 MPa, respectively55-56. As newly developed composites, these scaffolds could be exploited for bone tissue regeneration in human body. 3.4 Surface analysis of scaffolds



Figure 8a shows the cross-sectional SEM image of the lyophilized scaffolds morphology. Chemically crosslinked and non-crosslinked SA-CS, SA-CS-Col and SA-CS-Col-GO scaffolds showed porous structure. While non-crosslinked scaffolds showed wrinkled structures, the chemically crosslinked scaffolds showed no such wrinkled morphological features. The pore sizes of the scaffold were found to be between 75-250 µm which is more suitable for bone growth57. Pores in the scaffolds are crucial for cell migration, proliferation, vascularization and nutrient supply. Addition of Col and GO decreased the pore sizes of SA-CS scaffolds, while this reduction was more pronounced upon chemical crosslinking. That is, the pore sizes of noncrosslinked scaffolds SA-CS, SA-CS-Col, and SA-CS-Col-GO were in the range of 100-250 µm, 30-100 µm and 10–50 µm respectively. However, chemical crosslinking further decreased the pore sizes of these scaffolds to the range of 100-150 µm for SA-CS and 10-80 µm for SA-CSCol and SA-CS-Col-GO. Furthermore, chemical crosslinking of scaffolds (c-SA-CS-Col and cSA-CS-Col-GO) resulted in the loss of their inter-connective porous structures and the fibrous structure of collagen. . Figure 8b illustrates the percentage of porosity of the chemically crosslinked and non-crosslinked composite scaffolds, evaluated by micropore and macropore structures using the liquid displacement method. The non-crosslinked SA-CS, SA-CS-Col, and SA-CS-Col-GO scaffolds showed porosities 90%, 85% and 78%, respectively which showed a gradual decrease in the porous structures when higher crosslinking densities of scaffolds were achieved. Further, the crosslinked scaffolds exhibited reduced porosities: compared to their non-crosslinked counterparts, crosslinked SA-CS showed a significant decrease in porosity (62.7%) and the crosslinked scaffolds with Col and GO showed a dramatic reduction in porosity (20%).The crosslinked scaffolds showed compressed and contracted structures which corroborated the SEM


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image results. These variations in the porosity suggest differences in the mechanical properties of the scaffolds. In general, the degradation of biomedical implants provides breathing space for cells during tissue in growth and matrix deposition under in vivo conditions, which is crucial for engineering bone tissue. Owing to this reason, the controlling the scaffold degradation rate is considered critical in engineering bone tissue. The degradation rates of fabricated scaffolds were analyzed using PBS containing lysozyme solution (Figure 8c). Lysozyme is a primary enzyme in our body which plays a major role in vivo degradation. The degradation rate gradually increased with the increase in incubation time in lysozyme buffer solution for all the scaffolds. This result confirmed that the fabricated scaffolds were degradable nature in the presence of lysozyme solution. The weight loss of non-crosslinked SA-CS, SA-CS-Col, and SA-CS-Col-GO were 48.4±2.9, 33.2±2.3, and 27.8±2.5, respectively, after two weeks. The non-crosslinked SA-CSCol-GO showed significant decreasing in the degradation rate as compared with SA-CS and SACS-Col scaffold. In general, SA is an anionic polymer which possesses electrostatic interaction with the cationic polymer chitosan and collagen molecules to form a polyelectrolyte complex. These scaffolds are not stable in PBS for a long time. Calcium chloride enhances the stability of the scaffold by crosslinking through increasing electrostatic interactions of Ca2+ ions. Alternatively, incorporation of GO particles in alginate, chitosan, collagen solutions provides strong hydrogen bonding between GO and polymer molecules. Thus, hydroxyl, carboxyl, and epoxy groups on GO particle surface can react with amino, hydroxyl, carboxyl groups from alginate, chitosan, and collagen structure via strong hydrogen bonding. Hence, the SA-CS-ColGO scaffold showed lesser degradation rate compared to other non-crosslinked scaffolds. The weight losses of c-SA-CS, c-SA-CS-Col and c-SA-CS-Col-GO scaffolds were 31.3±2.3, 42±2.4



and 20±1.9, respectively, after two weeks. This result clearly showed that c-SA-CS reduced the degradation rate by calcium chloride crosslinking, as expected. Moreover, in c-SA-CS-Col and c-SA-CS-Col-GO the degradation rates after crosslinking with calcium ion were further reduced. When compared with non-crosslinked SA-CS-Col-GO, c-SA-CS-Col-GO scaffold showed less degradation. This is due to the hydrogen bonding network further increased by Ca ion, GO, SA, CS, and Col structure. 3.5 Swelling studies Swelling properties of scaffolds are very important for application in engineering bone tissues. Many natural polymers, such as SA and CS swell easily in the biological fluids. The initial swelling of the 3D scaffolds under in vitro and in vivo conditions is due to the increase in size of the pore which promotes the cell attachment and growth. The percent of swelling ratios vs time of immersion of non-crosslinked and crosslinked scaffolds in water and PBS are shown in Figure 9 (a-c). The photographic image in Figure 9a shows the swelling of the scaffolds before and after 2h in water and PBS. The swelling kinetics of scaffolds in water increased rapidly in about 1 h for all the samples and reached the equilibrium with slow rate of increment until 24 h(Figure 9b). In general, the scaffold´s water absorption capability might also be due to three-dimensional structure, microstructure, and hydrophilicity of molecules. The swelling ratio of crosslinked scaffolds was significantly lower when compared to non-crosslinked scaffolds, probably due to decreased levels of hydrophilic groups22. The difference in swelling ratio of SA-CS and SA-CSCol scaffold scan be attributed to their porous structures besides hydrophilic nature of Col and CS materials58. Presence of Col in CS enabled to retain the porous structure of the scaffolds with high elasticity and significant mechanical properties58. Further, incorporation of GO reduces the swelling ability of the scaffold by hydrogen bonding network among oxygenatedgroups59. The


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Ca2+crosslinked scaffolds exhibit further swelling resistance based on a stronger interaction between SA, metal ions and GO59. However, non-crosslinked scaffolds in PBS showed four-fold increase in swelling ratio compared to pure water due to diffusion of ions solution into scaffolds. Polyelectrolyte complex of SA-CS scaffold with macroporous structures exhibits superabsorbent property in PBS with rapid swelling ratio due to increasing the salt concentration, decrease of the osmotic pressure inside the scaffolds, while inter-polyelectrolyte complex stabilizes the scaffold through loop–ladder structural transition60. Sodium alginate-chitosan scaffold was dissolved after 3 h incubation in PBS, but SA-CS-Col and SA-CS-Col-GO scaffold maintained the disc shape in the 24 h swelling experiment. The swelling ratio was significantly decreased for SA-CS-Col-GO as compared to other non-crosslinked scaffolds, indicating that the incorporation of GO increased the crosslinking density of SA-CS-Col. The c-SA-CS scaffold showed reasonable increase in swelling ratio in PBS as compared to water(Figure 9c). Furthermore, the c-SA-CSCol and c-SA-CS-Col-GO scaffolds showed negligible swelling and reduced porosity, possibly due to strong interactions of calcium ions and oxygenated group of GO and polymer backbone with increased crosslinking density59. Here, the swelling ratio of fabricated scaffolds was examined using water (pH 7) and PBS (pH 7.4), since SA is highly sensitive to small changes in pH. The swelling ratio observed is higher in PBS compared to water and this could be due to the variation of pH which corroborated with our previous report61 and other literature reports62-63. 3.6 Cytocompatibility and osteogenesis study

Proliferation of MC3T3 osteoblast cells on TCPS surface, non-crosslinked and crosslinked 3D scaffolds was evaluated using MTT assay at three cell culture time intervals (1st, 3rd and 7th day) (Figure 10). The results showed increase in optical density (O.D) from day 1 to 7for TCPS



surface, crosslinked and non-crosslinked scaffolds which suggest an increased cell number. In non-crosslinked scaffolds, the number of cells on day 1 remained fairly constant compared to TCPS indicating that the incorporation of GO had no effect on the metabolic activity of cells. On day 3, cell population showed considerable increases for Col and GO incorporated scaffolds when compared to TCPS. The presence of Col molecules or GO surface with pendant functional groups enhanced cell attachment and proliferation64. Furthermore, on day 7, there was a threefold increase in cell proliferation when compared to day 1 cells due to controlled swelling behavior of scaffolds in culture media that facilitates more infusion of cells into the 3D porous interconnected structure as well as supplies more nutrients for cell growth. Similar to TCPS and non-crosslinked scaffolds, crosslinked scaffolds showed increased cell population with increase in culture time from day 1 to 7.Furthermore, the crosslinked scaffolds exhibited a significant increase in O.D for 3 and 7 day culture time points compared to TCPS and non-crosslinked scaffolds, suggesting increased number of cells. Compared to non-crosslinked scaffolds with no GO (SA-CS and SA-CS-Col), crosslinked scaffolds (c-SA-CS and c-SA-CS-Col) showed increased cell population at all time points of cell culture. This could be due to an increase in mechanical strengths of stable scaffolds following chemical crosslinking with calcium ions. However, cell proliferation on both non-crosslinked and crosslinked scaffolds bearing GO exhibited no major differences (Figure 10). These results suggested that the SA-CS-Col-GO scaffold is as suitable for osteoblast cell growth as chemically crosslinked scaffolds. However, in the case of crosslinked GO scaffold the metabolic activity of cells was inhibited when compared to non-crosslinked SA-CS-Col-GO scaffold, possibly due to reduction of pore size during chemical crosslinking process that could drastically reduce the infiltration of cells across the scaffolds. Hence, incorporation of polar GO in polymer matrix influenced the osteoblast cell-


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material interaction and favored osteoconductive surface for cell adhesion, growth and proliferation through its negatively charged surface64. Cell viability on scaffolds was determined using a live and dead assay kit which stains dead cells in red fluorescence and live cells in green fluorescence65. The fluorescent images of live and dead MC3T3 osteoblast cells cultured on non-crosslinked and crosslinked scaffolds for 1st, 3rd and 7th day cultures are shown in Figure 11. In presence of all the scaffolds, most of the cells were alive (green) along with a few dead (red) cells. The live cells appeared as bright green spots over the scaffolds as well distinguished spindle morphology. The ratio of dead versus live cells increased when cells were cultured for a more extended period. GO incorporated scaffold exhibited a higher number of live cells when compared to the other scaffolds, suggesting that the GO composites offered a better support for the osteoblast cell proliferation. However, only a minor difference was found between the total number of cells on the non-crosslinked and crosslinked GO scaffolds. In vitro osteogenic differentiation of MC3T3 osteoblast cells was confirmed by quantifying the mineral deposition using ARS assay. Figure 12 shows the calcium mineral deposition with and without MC3T3 cell in the osteogenic medium on TCPS surface, non-crosslinked and crosslinked scaffolds. Some mineralization was observed from the osteogenic medium without cells on TCPS and scaffolds at 14th day, as expected. However, this mineral deposition on TCPS and scaffolds by osteogenic medium was negligible. Analyses of the osteogenic differentiation of MC3T3 cells on TCPS and scaffolds revealed increased levels of ARS O.D. There was no significant difference in calcium mineral deposition on TCPS and SA-CS scaffold. However, the calcium mineral depositions increased with the addition of Col and GO compared to TCPS and SA-CS which correlate well with the increased levels of ARS O.D. (Figure 10).These results



suggest that Col and GO support osteogenic differentiation of MC3T3 cells. The crosslinked scaffolds SA-CS and SA-CS-Col showed significantly higher mineralization than TCPS, noncrosslinked SA-CS, and SA-CS-Col scaffolds. The GO incorporated scaffolds showed higher mineral deposition in both non-crosslinked and crosslinked scaffolds when compared to scaffolds with no GO and TCPS surface. Further, there was no significant difference in the mineral deposition on SA-CS-Col-GO and c-SA-CS-Col-GO, suggesting that the chemical crosslinking of GO scaffolds did not affect the mineralization. Furthermore, the incorporation of GO in polymer matrix increased the ionic crosslinking between polymer chain and GO particles. The GO particles in polymer matrix act like a Ca2+ion during chemical crosslinking process. Thus, the GO incorporated scaffolds were found to induce osteogenesis, leading to calcium mineral deposition, suggesting that these novel scaffolds are potential candidates for biomedical implants. 4.

Conclusions Bone tissue engineering is an emerging new direction for the healing of defected and

cancerous bone. Three different scaffolds namely SA-CS, SA-CS-Col and SA-CS-Col-GO were successfully prepared by freeze-drying technique followed by ionically crosslinking with calcium ions. Our results demonstrated that integration of GO significantly influenced the morphology, swelling ability and defined mechanical property of scaffolds. Further, collagen fiber was not affected by the addition of GO in the non-crosslinked composites scaffolds which was confirmed by SEM. Our results suggest that SA-CS, SA-CS-Col and novel SA-CS-Col-GO scaffolds are potential candidates to support osteoblast cell growth and osteogenic differentiation. More specifically, the novel SA-CS-Col-GO scaffolds demonstrated an added advantage in promoting osteoblast cell growth and osteogenic differentiation. Importantly, our


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studies show that the SA-CS-Col-GO scaffolds have an outstanding biocompatibility, controlled degradation rate, and sufficient mechanical properties to serve as novel scaffolds for engineering bone tissue. ASSOCIATED CONTENT Supporting Information The synthesized GO particle was dispersed in deionized water for SEM and DLS analysis. (a) SEM image, (b) the particle size distribution curve and (c) the zeta potential curve of GO in dispersion state (Figure S1a-c); In vitro biodegradation study on non-crosslinked and chemically crosslinked scaffolds in PBS containing lysozyme (10,000 U/L) at 37 ºC. The photographic image shows the dried scaffolds at Day 0 (before) and Day14 (after) degradation (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org/. Acknowledgements KE acknowledge for the postdoctoral fellowship at IISc, Bangalore (Indian Institute of Science) funded by DST (Department of Science and Technology) India and University of Sao Paulo (USP), Sao Paulo funded by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) Brazil, Grant file number 2011/21442-6 and 2015/19694-8. References (1) O brien, F. J. Biomaterials & Scaffolds for Tissue Engineering. Materials today 2011, 14 (3), 88-95. (2) Langer, R. Biomaterials in Drug Delivery and Tissue Engineering: One Laboratory's Experience. Accounts of Chemical Research 2000, 33 (2), 94-101. (3) Kuo, Z.-K.; Lai, P.-L.; Toh, E. K.-W.; Weng, C.-H.; Tseng, H.-W.; Chang, P.-Z.; Chen, C.C.; Cheng, C.-M. Osteogenic Differentiation of Preosteoblasts on A Hemostatic Gelatin Sponge. Scientific reports 2016, 6, 32884. (4) Zhang, D.; Wu, X.; Chen, J.; Lin, K. The Development of Collagen Based Composite Scaffolds for Bone Regeneration. Bioactive Materials 2017, DOI: 10.1016/j.bioactmat.2017.08.004. (5) Fu, C.; Bai, H.; Hu, Q.; Gao, T.; Bai, Y. Enhanced Proliferation and Osteogenic Differentiation of MC3T3-E1 Pre-Osteoblasts on Graphene Oxide-Impregnated PLGA–Gelatin Nanocomposite Fibrous Membranes. RSC Advances 2017, 7 (15), 8886-8897. (6) Maisani, M.; Pezzoli, D.; Chassande, O.; Mantovani, D. Cellularizing Hydrogel-Based Scaffolds to Repair Bone Tissue: How to Create A Physiologically Relevant MicroEnvironment? Journal of Tissue Engineering 2017, 8, 2041731417712073.



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(53) Chen, Z.; Wei, B.; Mo, X.; Lim, C.; Ramakrishna, S.; Cui, F. Mechanical Properties of Electrospun Collagen–Chitosan Complex Single Fibers and Membrane. Materials Science and Engineering: C 2009, 29 (8), 2428-2435. (54) Kuo, C. K.; Ma, P. X. Ionically Crosslinked Alginate Hydrogels as Scaffolds For Tissue Engineering: Part 1. Structure, Gelation Rate and Mechanical Properties. Biomaterials 2001, 22 (6), 511-521. (55) Ha, S. K. Finite Element Modeling of Multi-Level Cervical Spinal Segments (C3–C6) and Biomechanical Analysis of an Elastomer-Type Prosthetic Disc. Medical engineering & physics 2006, 28 (6), 534-541. (56) Gosline, J.; Lillie, M.; Carrington, E.; Guerette, P.; Ortlepp, C.; Savage, K. Elastic Proteins: Biological Roles and Mechanical Properties. Philosophical Transactions of the Royal Society of London B: Biological Sciences 2002, 357 (1418), 121-132. (57) Cheung, H.-Y.; Lau, K.-T.; Lu, T.-P.; Hui, D. A Critical Review on Polymer-Based BioEngineered Materials for Scaffold Development. Composites Part B: Engineering 2007, 38 (3), 291-300. (58) Yan, L. P.; Wang, Y. J.; Ren, L.; Wu, G.; Caridade, S. G.; Fan, J. B.; Wang, L. Y.; Ji, P. H.; Oliveira, J. M.; Oliveira, J. T. Genipin‐Cross‐Linked Collagen/Chitosan Biomimetic Scaffolds for Articular Cartilage Tissue Engineering Applications. Journal of Biomedical Materials Research Part A 2010, 95 (2), 465-475. (59) Vilcinskas, K.; Zlopasa, J.; Jansen, K.; Mulder, F. M.; Picken, S. J.; Koper, G. J. Water Sorption and Diffusion in (Reduced) Graphene Oxide‐Alginate Biopolymer Nanocomposites. Macromolecular Materials and Engineering 2016, 301 (9), 1049-1063. (60) Yu, S. H.; Mi, F. L.; Wu, Y. B.; Peng, C. K.; Shyu, S. S.; Huang, R. N. Antibacterial Activity of Chitosan–Alginate Sponges Incorporating Silver Sulfadiazine: Effect of Ladder‐Loop Transition of Interpolyelectrolyte Complex and Ionic Crosslinking on the Antibiotic Release. Journal of applied polymer science 2005, 98 (2), 538-549. (61) Khajuria, D. K.; Mahapatra, D. R. In Photonic Hydrogel Beads for Controlled Release of Risedronate, Photonic Therapeutics and Diagnostics X, International Society for Optics and Photonics: 2014; p 892640. (62) Park, H.; Guo, X.; Temenoff, J. S.; Tabata, Y.; Caplan, A. I.; Kasper, F. K.; Mikos, A. G. Effect of Swelling Ratio of Injectable Hydrogel Composites on Chondrogenic Differentiation of Encapsulated Rabbit Marrow Mesenchymal Stem Cells In Vitro. Biomacromolecules 2009, 10 (3), 541-546. (63) Gupta, N. V.; Shivakumar, H. Investigation of Swelling Behavior and Mechanical Properties of A Ph-Sensitive Superporous Hydrogel Composite. Iranian journal of pharmaceutical research: IJPR 2012, 11 (2), 481. (64) Depan, D.; Girase, B.; Shah, J.; Misra, R. Structure–Process–Property Relationship of the Polar Graphene Oxide-Mediated Cellular Response and Stimulated Growth of Osteoblasts on Hybrid Chitosan Network Structure Nanocomposite Scaffolds. Acta biomaterialia 2011, 7 (9), 3432-3445. (65) Kolanthai, E.; Colon, V. S. D.; Sindu, P. A.; Chandra, V. S.; Karthikeyan, K.; Babu, M. S.; Sundaram, S. M.; Palanichamy, M.; Kalkura, S. N. Effect of Solvent; Enhancing the Wettability and Engineering the Porous Structure of A Calcium Phosphate/Agarose Composite for Drug Delivery. RSC Advances 2015, 5 (24), 18301-18311.



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List of Table Table 1: Compressive modulus of non-crosslinked and chemically crosslinked scaffolds. List of Figures Figure 1: Schematic representation of the synthesis of nanoparticles graphene oxide using graphite flakes. The figure illustrates the preparation of graphene oxide-based SA-CS-Col porous structure scaffolds with and without chemical crosslinking by freeze-drying technique and their potential application for bone tissue engineering using MC3T3 cells as a model system. Figure 2: Photographic images of non-crosslinked and chemically crosslinked scaffolds prepared by freeze-drying technique. Figure 3(a): XRD patterns of graphite and GO, (b) FTIR spectrum of GO, (c) Raman spectrum of GO and (d) SEM image of GO. Figure 4: FTIR spectra of polymer powders, non-crosslinked and chemically crosslinked scaffolds (a) SA, (b) CS, (c) Col, (d) SA-CS, (e) SA-CS-Col, (f) SA-CS-ColGO, (g) c-SA-CS, (h) c-SA-CS-Col, and (i) c-SA-CS-Col-GO. Figure 5: Schematic representation of possible hydrogen bond formation between SA, CS, Col molecules and GO particle.(a) Chemically non-crosslinked SA-CS-Col-GO scaffold, (b) Chemically crosslinked SA-CS-Col-GO scaffold. Figure 6: XRD patterns of newly developed non-crosslinked and chemically crosslinked scaffolds. Figure 7: Typical compressive stress-strain curves of non-crosslinked SA-CS, SA-CS-Col, SA-CS-Col-GO

and

chemically

crosslinked

c-SA-CS,

Col-GO scaffolds.


c-SA-CS-Col,

c-SA-CS-

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Figure 8: Studies on the porous structure of scaffolds.SEM images showing porous structure of (a) non-crosslinked and chemically crosslinked scaffolds, (b) percentage porosity in noncrosslinked and chemically crosslinked scaffolds estimated by liquid displacement method and (c) Crosslinked and non-crosslinked scaffold biodegradation in vitro by lysozyme. Experiments were performed at 37 ºC in PBS for two weeks in triplicate. Statistically significant (p< 0.05) difference compared to SA-CS, SA-CS-Col, SA-CS-Col-GO, c-SA-CS, c-SA-CS-Col, and c-SACS-Col-GO are indicated by #, ∆, Φ, δ, and Ψ, respectively. Figure 9: Studies on the swelling behavior of scaffolds.(a)Photographic images of scaffolds before and after allowing to swollen for 2 h. Note that after 2h incubation, SA-CS scaffold was completely dissolved in PBS solution. (b and c)show swelling ratio of the scaffolds in water and PBS. Figure 10:Differential effects of scaffolds on cell proliferation.MC3T3 cell proliferation on polystyrene (TCPS) surface, non-crosslinked and crosslinked scaffolds was measured using MTT assay at 1, 3 and 7 days of culturing. Statistically significant (p< 0.05) difference compared to TCPS surface, SA-CS, SA-CS-Col, SA-CS-Col-GO, c-SA-CS, c-SA-CS-Col, and c-SA-CSCol-GO are indicated by *, #, ∆,Φ, δ, and Ψ, respectively. Figure 11:Analysis of live and dead cells on scaffolds by fluorescence staining.MC3T3 cells grown on the surface of polystyrene (TCPS) plate, non-crosslinked and chemically crosslinked scaffolds were used to stain live (green) and dead (red) cells after culturing for 1, 3 and 7 days. Scale bar represents 100 µm. Figure 12: Differential effects of scaffolds on mineralization. The calcium mineral deposition by MC3T3 cells grown on TCPS surface and various scaffolds were measured using Alizarin Red S (ARS).A low-level mineralization was observed for cells grown on polystyrene tissue culture



plate in osteogenic supplemented growth medium. The absorbance plot for retained dye from mineralized sample was measured at 415 nm on 14thday.Statistically significant (p< 0.05) difference compared to polystyrene (TCPS) surface, SA-CS, SA-CS-Col, SA-CS-Col-GO, c-SACS, c-SA-CS-Col, and c-SA-CS-Col-GO are indicated by *, #, ∆, Φ, δ, and Ψ, respectively.


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Table 1

Compression modulus (MPa) Samples code Strain 10 %

Strain 20 %

Strain 40 %

SA-CS

0.11±0.06

0.25±0.11

0.52±0.34

SA-CS-Col

0.41±0.25

0.54±0.56

0.66±0.20

SA-CS-Col-GO

0.51±0.01

0.62±0.11

0.87±0.05

c-SA-CS

0.21±0.01

0.30±0.02

0.81±0.01

c-SA-CS-Col

0.61±0.04

0.62±0.18

2.52±1.13

c-SA-CS-Col-GO

0.91 ±0.49

1.16±0.410

5.33±1.54



Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5


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Figure 6



Figure 7


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Figure 8



Figure 9


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Figure 10



Figure 11


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Figure 12



TOC figure


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