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Original Article
DOI: 10.1016/j.jmrt.2019.08.043
Open Access
Available online 18 September 2019
Application of supercritical gel drying method on fabrication of mechanically improved and biologically safe three-component scaffold composed of graphene oxide/chitosan/hydroxyapatite and characterization studies
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Pelin Yılmaza, Elif öztürk Era, Sezgin Bakırdereb, Kutlu ülgenc, Belma özbeka,
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bozbek@yildiz.edu.tr

Corresponding author.
a Yildiz Technical University, Chemical Engineering Department, Davutpasa Campus, Esenler/İstanbul, 34220, Turkey
b Yildiz Technical University, Chemistry Department, Davutpasa Campus, Esenler/İstanbul, 34220, Turkey
c BoĿaziçi University, Chemical Engineering Department, Bebek/İstanbul, 34342, Turkey
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Tables (4)
Table 1. Compositional analysis of synthesized HAp obtained from EDS analysis.
Table 2. Component ratio of scaffolds produced and their relative cell viability values.
Table 3. Surface area and porosity analysis of scaffold D.
Table 4. Recent studies on multi-component scaffolds for bone tissue engineering applications.
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Abstract

Multicomponent-porous scaffolds have recently gained much attention in bone tissue engineering applications due to their ability to mimic the composite structure of natural bone tissue. In the present study, it was aimed to fabricate biologically safe and mechanically improved three-component scaffolds for bone tissue engineering applications. This is the first original report on the application of supercritical gel drying for the fabrication of three-component scaffolds composed of graphene oxide (GO) synthesized by Improved Hummers Method, chitosan (CS) and hydroxyapatite (HAp) derived from eggshells. The phase, morphology, mechanical property and in-vitro biocompatibility of scaffolds were investigated by FTIR, XRD, SEM, TEM, BET, TGA, Universal Instron Mechanical Test System, and MTT testing. For the preparation of scaffolds, GO, CS and HAp solutions were blended at various ratios. Resulting mixtures were molded, frozen, exposed to water-acetone substitution procedure, and dried by supercritical gel drying, respectively. Then, dried scaffolds were subjected to MTT testing for cytotoxicity analysis to examine toxicity effect of GO. Results revealed dose-dependent cytotoxicity effect of GO on MC3T3-E1 cell line. The highest relative cell viability was observed with three-component scaffold composed of GO:1wt%, CS:39wt% and HAp:60wt%. According to characterization studies, this original report demonstrated that scaffold produced with the mentioned procedure had a three-dimensional porous sponge-like structure, highest relative cell viability, and increased mechanical compressive strength compared to other scaffolds fabricated. These properties enable scaffold with combined improvement in biological and mechanical properties to be a promising candidate for application in bone tissue materials.

Keywords:
Hydroxyapatite
Graphene oxide
Chitosan
Three-component scaffold
Supercritical gel drying
Characterization studies
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1Introduction

Bone is a large connective tissue, which is responsible for locomotion, support, and protection of soft tissues. It is calcium and phosphate reservoir which provides structural integrity of the body [1,2]. It has a natural composite structure composed of organic materials (mainly collagens), and inorganic materials (nanocrystalline hydroxyapatite (HAp)) and water [2⿿5]. Collagen is an agent which can modulate adhesion of osteoblasts and fibroblasts since it has natural binding sites recognized by osteoblast and fibroblast cells. On the other hand, HAp is a source of calcium and phosphate ions for bone cells [6].

The increase in aging rate, obesity, and poor physical activity lead to bone disorders in human beings worldwide. Although bone tissue has a high capacity of regeneration [7⿿9], complicated large bone defects cannot be regenerated via normal physiological processes. In this case, additional treatments are required for proper regeneration such as intervention in the forms of bone grafts and etc. Autografts and allografts can be listed as two conventional ways for bone grafting [10]. Autografts are extracted from the patients themselves while allografts are extracted from cadavers, and need to be sterilized [9,11]. Another alternative way for bone tissue regeneration is transplantation which is conventionally applied to treat bone defects. However, to eliminate the rejection of transplanted tissue, the patients must take immunosuppressants for the rest of their lives [12]. Since the methods aforementioned have several disadvantages, the ideal way to compete with undesirable consequences is to use tissue engineering biomaterials which can mimic the natural environment of cells and stimulate them for cell growth, differentiation, migration and finally regeneration of new bone tissues. Bone tissue engineering strategy, associated with the regeneration of diseased or damaged bone tissues by controlling biological microenvironment using proper biomaterials, arises as a promising alternative to conventional methods. Regarding to this strategy, the mechanical, structural and biological functionality properties of scaffolds produced depend on the selection of biomaterials and fabrication methods.

Hydroxyapatite (HAp) is one of the calcium phosphate (CP) based biomaterial with a Ca/P molar ratio of 1.67. HAp is an inorganic constituent of bones and teeth [13]. Therefore, HAp is an attractive and necessary material used for the production of bone tissue engineering scaffolds due to its similar properties with natural bone such as structure, bioactivity, biocompatibility, and osteoconductivity [14⿿18]. However, the weak mechanical properties of hydroxyapatite; specifically low tensile strength, brittleness, and poor impact resistance which restrict its unique usage in bone tissue engineering applications [16].

Graphene oxide (GO) is defined as single-atomic-layered material composed of carbon, hydrogen, and oxygen molecules, and produced by oxidation of graphite crystals [19]. GO has significant chemical, optical and electrical properties, which enable GO to be used in various applications [20]. Hence, GO is preferred as an additive material used for the production of bone tissue engineering scaffolds to improve their properties such as mechanical strength, cell adhesion, differentiation and proliferation [21,22].

Chitosan (CS) is a natural cationic biopolymer and structurally similar to glycosaminoglycans which are major constituents of natural extracellular matrix (ECM) [23,24]. For the production of bone tissue engineering scaffolds, chitosan is preferred due to its attractive properties such as biocompatibility, biodegradability, and non-toxicity. Additionally, the hydrophilic surface of chitosan promotes improvements in cell adhesion, proliferation, differentiation and mineralization [25]. However, the mechanical properties of chitosan should be enhanced for its utilization in bone tissue engineering applications as its structure is very stiff and brittle [24]. For enhancement of the mechanical properties of chitosan, crosslinking agents and various biomaterials are reinforced into its structure to obtain a similar structure with natural ECM [26,27].

The supercritical gel drying method is frequently used to dry 3D scaffolds for maintenance of macro- and nano-structure of the hydrogel as this drying method does not cause any formation of vapor-liquid transition and surface tensions in hydrogel pores [28]. For the application of this technique, supercritical carbon dioxide (SC⿿CO2) is preferred due to its properties such as high affinity with almost all organic solvents, low toxicity, and availability. However, this method cannot be directly applied to polymeric hydrogels since water has limited solubility in supercritical conditions of carbon dioxide. This case prevents SC⿿CO2 affinity for water, thus water inside of polymeric hydrogel cannot be removed. This case results in failure for the drying of polymeric hydrogels. To overcome this failure, water should be substituted with an organic solvent such as acetone or ethanol. In this way, an organic solvent can be removed by SC⿿CO2 for completion of the drying process with success [29].

Recently, multicomponent porous scaffolds have gained attention in bone tissue engineering applications since they have a high ability to mimic the composite structure of natural bone tissue. The multi-component scaffolds have better compatibility with natural bone tissue, as their composite structures composed of inorganic and organic materials, as stated in the several studies; for example, hydroxyapatite-chitosan composite scaffolds have been combined with various components such as; carbon nanotube [30], chondroitin sulphate [31], amylopectin [31], collagen [32], graphene oxide [33], β-tricalciumphosphate (β-TCP) [34], alginate [35], gelatin-alginate [36], nanodiopside [37], reduced graphene oxide [38], dextran [39], silica [40], zein [41], collagen-functionalized multiwalled carbon nanotube [42] and graphene oxide [43] for fabrication of bone tissue engineering scaffolds with desired properties.

In the present study, biomaterial selection for production of scaffolds is inspired by the natural bone tissue constituents. For this purpose, three-component scaffolds composed of graphene oxide/chitosan/hydroxyapatite-(GO/CS/HAp) was aimed to produce to be used for bone tissue engineering applications. Although, there are many studies performed on the production of bone tissue engineering scaffolds, to the best of our knowledge, this is the first original report on the fabrication of three-component scaffold, composed of graphene oxide which synthesized by Improved Hummers Method, chitosan (purchased) and hydroxyapatite which is derived from eggshells, with application of supercritical gel drying method. For this purpose, the scaffolds were prepared by blending of GO, CS and HAp solutions at various ratios. The resulting scaffold mixtures were frozen following molding stages. After applying water-acetone substitution process to the scaffolds prepared, they were subjected to the supercritical gel drying procedure. Besides, the possible cytotoxicity effects of GO added at various ratios into scaffold composites were examined by in-vitro cell viability analysis, MTT testing. After evaluation of the data, scaffold with the highest cell viability was chosen for further characterization studies by FTIR, XRD, TGA, SEM, TEM, BET and mechanical compressive strength analyses for determination of its usability in bone tissue materials. The novelty of this work is to apply the supercritical gel drying method on the fabrication of mechanically improved and biologically safe three-component scaffold composite structures, composed of graphene oxide, chitosan, and hydroxyapatite, to be used in bone tissue engineering applications.

2Materials and methods2.1Materials

Graphite powders with high purity value to 99.9995% were purchased from Alfa Aesar. Chitosan was supplied from Acros firm with 9012-76-4 CAS number. Glutaraldehyde used as a crosslinking agent, (50% aqueous solution), was supplied from AppliChem firm. Potassium permanganate (KMnO4), hydrogen peroxide solution (35%) (H2O2), hydrochloric acid (37%) (HCl), ortho-Phosphoric acid (85%) (o-H3PO4), sulfuric acid (95⿿98%) (H2SO4), acetone and ammonia solution (25%) (NH4OH) were supplied from Merck.

2.2Synthesis of HAp

For hydroxyapatite synthesis by wet chemical precipitation method, calcium oxide, which is derived from eggshells, and ortho-phosphoric acid (85%) were used as calcium and phosphate precursors, respectively [44]. Firstly, eggshells were washed with an excess amount of distilled water, boiled in distilled water, and then, removed their inside membranes. The eggshells were dried at 100°C for 12h to eliminate odor and any volatile contaminants and ground using a grinder. Then, eggshell powder, which is mostly composed of calcium carbonate, was calcined in a laboratory furnace at temperature of 900°C with 10°C heat rate for 2h and transformed to calcium oxide. This calcium oxide was dissolved in distilled water to get 1M calcium hydroxide solution. Calcium hydroxide solution was heated up to 40°C and stirred at 600rpm using a magnetic stirrer. Then, 0.6M of phosphoric acid solution was transferred to calcium oxide solution using a dropper. The pH value of the resultant mixture was adjusted to 9 by adding ammonium hydroxide solution (25%, v/v). The final mixture was stirred for further 2h. The formation of precipitation was completed by keeping the mixture for 24h at room temperature. After that, the precipitate was washed for three times with 200mL distilled water. Then, the solid part was separated by centrifugation at 6000rpm for 30min. The washed solid part was transferred into a petri dish, and then dried at 100°C for 12h. Then, the dried solid part (hydroxyapatite powder) was ground using a mortar, and pestle to produce a fine powder. Finally, the hydroxyapatite was calcined in the laboratory furnace at 900°C with 10°C/min heating rate for 2h to increase the crystallinity degree of HAp product.

2.3Synthesis of GO

Graphene oxide was synthesized by Improved Hummers Method which was introduced by Marcano et al. [45]. The oxidation reaction of graphite was performed with KMnO4 in a mixture of H2SO4/H3PO4 (9:1 v/v) for production of GO with a higher extent of oxidation [45]. Graphite powder (3g) and KMnO4 (9g) were added into oxidation medium including 360mL of H2SO4 and 40mL of H3PO4. The reaction medium was then heated up to 50°C and stirred for 12h at 150rpm. Afterward, the reaction medium was cooled down to room temperature and poured into the ice with 3mL of H2O2 30%. This mixture was centrifuged at 6000rpm for 30min for removal of the liquid part. The solid part was washed in succession using 200mL of distilled water, 5N 200mL HCl and 200mL of ethanol. Then, the solid part was separated from the liquid part using centrifugation. Finally, the centrifuged solid part was transferred into a petri dish and dried at 50°C for 18h.

2.4Preparation of scaffolds

For the preparation of the scaffolds; graphene oxide/chitosan, chitosan/hydroxyapatite and graphene oxide/chitosan/hydroxyapatite; GO, CS and HAp solutions were blended at various ratios to produce scaffolds in various compositions. Glutaraldehyde was used as a crosslinking agent for the preparation of scaffolds. Otherwise, water-acetone substitution, which is the necessary step for supercritical gel drying, could not be achieved due to the collapsing tendency of the scaffold structure. The steps for preparation of the scaffolds were explained in details and given as below:

Preparation of chitosan solution: Chitosan was weighed and dissolved in 2% (v/v) acetic acid solution to reach 4% (w/v) concentration, and this mixture was stirred for 2h at 45°C.

Preparation of graphene oxide/chitosan (GO/CS) scaffolds: Graphene oxide was weighted in defined amount and dissolved in distilled water by sonication for 2h. The homogeneous graphene oxide solution was transferred to chitosan solution (4%, w/v) by using a dropper. This mixture was stirred for 1h at 40°C, and 1% (v/v) of glutaraldehyde solution for its action as a crosslinking agent was added into this solution. Then, the final mixture was stirred for additional 2h at 40°C. The resultant solution was transferred to 21 well plates, and they were frozen at ⿿18°C for 24h for the formation of the hydrogels. Then, these hydrogels were put an acetone bath at ⿿18°C for 24h for water-acetone substitution stage. After that, these samples were subjected to dry based on the supercritical gel method. Unless otherwise stated, the same procedure for stages of molding, freezing, water-acetone substitution and drying was followed, respectively, for preparation of each scaffold.

Preparation of chitosan/hydroxyapatite (CS/HAp) scaffolds: Hydroxyapatite was weighted in defined amount and dissolved in distilled water by sonication for 2h. Hydroxyapatite solution was transferred to chitosan solution (4%, w/v) by using a dropper. This mixture was stirred for 1h at 40°C, and 1% (v/v) of glutaraldehyde solution for its action as a crosslinking agent, and was added to this solution, and then stirred for additional 2h at 40°C. After that, the resultant homogenous solution was molded, frozen, and then dried, respectively.

Preparation of graphene oxide/chitosan/hydroxyapatite (GO/CS/HAp) scaffolds: Firstly, graphene oxide solution at a defined amount, was transferred to chitosan solution (4% w/v), and this mixture was stirred for 1h at 40°C. Secondly, the hydroxyapatite solution was transferred to the graphene oxide-chitosan solution, and this mixture was stirred at 500rpm for 2h at 40°C. Following this step, the crosslinking agent, glutaraldehyde of 1% (v/v), was added to this mixture and stirred for an additional 2h at 40°C. The resulting homogenous solution was molded, frozen, and then dried, respectively. To investigate the toxicity effect of GO on MC3T3-E1 cell lines, the scaffolds were prepared at various GO ratios (0.5, 1, 2 and 4wt%). The same procedure for stages of molding, freezing, and drying was followed for the preparation of each scaffold with GO at various ratios.

2.5Drying procedure of prepared scaffolds

Supercritical gel drying was carried out in the following procedure: after the water-acetone substitution, samples were put in a high-pressure vessel filled from the top with SC⿿CO2. The drying procedure was performed at a constant flow rate of SC⿿CO2 (1kg/h), the temperature of 70°C and pressure of 200bar for 4h. After depressurization of the system, produced scaffolds were characterized in terms of their chemical, biological and mechanical compressive strength properties.

In the present study, all the experiments were carried out at least in duplicate and the data reported were the average of the measurements. The reproducibility between trials was within the range of ±5%.

2.6Characterization studies

UV-Visible Spectrophotometer: The double beam UV-1800 SHIMADZU spectrophotometer, which has a 190⿿1100nm spectral range with ±0.3nm wavelength accuracy, was used to determine the UV⿿vis spectrum of aqueous solutions.

Raman Spectroscopy: The Raman spectra of samples were recorded at 785nm laser excitation using Perkin Elmer Raman Station 400F instrument.

Fourier Transform Infrared (FTIR) Spectrometer: Bruker Tensor 27 Attenuated Total Reflection (ATR) FTIR Spectrometer, which has ±0.01cm⿿1 wavenumber accuracy and photometric accuracy is ±0.1% T, was used to determine the chemical structure of the components and produced scaffolds. The analyses were performed at room temperature and specific wavelength range (450⿿4000cm⿿1 range) to determine the functional groups of the samples.

X-Ray Powder Diffraction (XRD) Analysis: PANalytical X⿿Pert Pro Analyzer was used to identify the phase of the synthesized components and scaffolds. Cu-Kα radiation source was used for XRD analyzer, and the scanning angle 2θ can be arranged from 2° to 90° with a step scanning rate of 3°min⿿1 at 45kV and 40mA. The crystallinity size of the samples was calculated using the Scherer formula as given in Eq. (2.1):

In Eq. (2.1), Dp is the average particle size of the powder, k is the constant (0.9), λ is the X-ray emission wavelength (Cu=1.54 A), b is the half-width of the diffraction line (002) for the reference of polycrystalline sample, and θ represents the Bragg⿿s angle of the diffraction line [46].

Thermogravimetric/Differential Thermal Analyzer (TG/DTA): EXSTAR SII TG/DTA6300 Analyzer, was used to determine the thermal behavior of the components and scaffolds produced. In the present study, the heating rate was arranged as 10°C/min, and the analyses were performed at the temperature range of 25⿿1000°C under the nitrogen atmosphere.

Scanning Electron Microscope (SEM): Zeiss EVO LS10 Scanning Electron Microscope operating in beam mode at 20kV with a secondary electron detector was used to characterize the morphological structure of produced scaffolds and hydroxyapatite. The solid samples were coated by gold-palladium for 45s in argon plasma with Quorum SC7620 Sputter Coater.

Transmission Electron Microscopy (TEM): Transmission electron images of the samples were recorded on JEOL JEM 2100 HRTEM operating at 200kV. Gatan Model 833 Orius SC200D CCD Camera was used to take the images. TEM grids (Electron Microscopy Sciences, CF200-Cu, 200 mesh) were used in the instrument.

Brunauer-Emmett-Teller (BET) Surface Area Analysis: The specific surface area of produced scaffolds were analyzed using Quantachrome Quadrosorb SI at 77K. For the measurements, the samples were degassed at the temperature of 393K for 3h, and nitrogen gas was used as an adsorbate gas.

Cell Viability Analysis: The cytotoxicity analysis of produced scaffolds named as A, B, C, D, E, and F were performed using MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) assay. This analysis was carried out to investigate the cell viability and proliferation on produced scaffolds. For this purpose, MC3T3-E1 cells were incubated at 37°C in a moist environment of 5% CO2. Then, the medium was removed, and the cells were washed with PBS (phosphate-buffered saline). Afterward, the cells were plated on 24 well plates with the scaffolds (three samples were used for each scaffold produced) in α-MEM culture media with 10% FBS (v/v), 1% penicillin-streptomycin (v/v) at a density of 1ÿ105cells/mL, and the plates were incubated at 37°C in 5% of CO2 to investigate the cytotoxic and proliferative effects. The cells plated free from scaffold were used as a negative control, and the cells treated with DMSO (dimethyl sulfoxide) were used as a positive control. Afterward, 50μL MTT solution was added to each well and incubated for 3h at 37°C. The formation of formazan crystals was obtained due to the reduction of MTT by viable cells. Then, 500μL DMSO was added to dissolve the formazan crystals. After 30min, the absorbance values of the plates were measured at 570nm with Elisa Reader. The recorded absorbance value is proportional to the amount of viable cells. The results were expressed by comparing the absorbance values of the cell-scaffold (ASample) construct with the control groups. The negative control group absorbance (ANegative control) was standardized as 100% viability, and the relative cell viability (RCV%) was calculated according to the Eq. (2.2) given as below.

Mechanical Compressive Strength Analysis: The cylindrical samples with a diameter of 1cm and a high of 1cm were mechanically tested by a Universal Instron Mechanical Testing System (Instron 5982). The crosshead speed of the instrument was adjusted to 0.5mm/min, and each measurement was repeated three times, and the average was taken for each set of the data. Then, the mechanical compressive strength values were calculated by dividing the maximum load (N) to the cross-sectional area of produced scaffolds.

3Results and discussions3.1Characterization studies of eggshells3.1.1Fourier transform infrared spectroscopy (FTIR) analysis

FTIR analyses of eggshell powder (calcium carbonate) and calcined eggshell powder (calcium oxide) were represented in Fig. 1. The FTIR spectrum of eggshell powder (calcium carbonate) indicated that the peak observed at 1796cm⿿1 corresponded to combination modes of different CO32⿿ as can be seen in Fig. 1. The characteristic peaks of carbonate group in CaCO3 were observed at 1393, 873 and 712cm⿿1[44]. The FTIR spectrum of calcined eggshell powder given in Fig. 1 confirmed that the successful transformation of CaCO3 to CaO. The bands at 1413 and 875cm⿿1 were associated with CO stretching, and OH groups bound with calcium atoms were observed at 3647cm⿿1[44]. It was concluded that the calcination process provided an increase in the presence of CaO [47].

Fig. 1.

FTIR spectra of eggshell powder and calcined eggshell powder.

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3.1.2X-ray powder diffraction (XRD) analysis

The XRD patterns of crushed and calcined eggshell samples were given in Fig. 2. The major and intense peak appeared at 2θ values of 29.51° was attributed to the presence of CaCO3. After the calcination step, it was clearly seen that the intensity of the peak observed at 2θ values of 29.51° decreased due to the transformation of calcium carbonate to calcium oxide. In Fig. 2, the diffraction peaks obtained within 2θ values of 28.88°, 34.37° and 54.53° corresponded to the presence of calcium oxide phase. The diffraction peaks within 2θ values of 18.29°, 47.48°, and 51.11° were attributed to the presence of Ca(OH)2 phase [48].

Fig. 2.

XRD patterns of eggshell powder and calcined eggshell powder.

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3.2Characterization studies of HAp3.2.1Scanning electron microscopy (SEM) analysis

The agglomeration tendency of HAp could be clearly seen from the SEM images of synthesized HAp at 5.00 KX and 50.00 KX magnifications (Fig. 3).

Fig. 3.

SEM images of HAp A) at 5.00 KX and B) at 50.00 KX.

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3.2.2Energy dispersive X-ray spectroscopy (EDS) analysis

EDS was used to analysis of the elemental composition of HAp. The presence of Ca, P and O elements in the sample was clearly seen in EDS pattern of HAp (Fig. 4). The negligible amount of Au and Pd elements were also detected in the EDS pattern of HAp due to the coating of HAp for SEM analysis. According to the elemental composition of HAp given in Table 1, the molar ratio of Ca/P was found as 1.68, which is a similar value with pure hydroxyapatite found in bone tissue [49].

Fig. 4.

EDS pattern of Hap.

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Table 1.

Compositional analysis of synthesized HAp obtained from EDS analysis.

O (wt%)  P (wt%)  Au (wt%)  Pd (wt%)  Ca (wt%) 
29.8  17.4  12.2  2.8  37.8 
3.3Characterization studies of GO3.3.1UV⿿vis spectroscopy analysis

The UV⿿vis spectrum of aqueous GO measured at 200⿿800nm wavelength range was in agreement with the characteristic sharp absorption peak at ⿼228nm and broad shoulder at ⿼300nm as seen in Fig. 5[50]. The broad shoulder was assigned to n⿿Ͽ* transitions of precedence of epoxide (COC) and peroxide (ROOR) like linkages [45]. The sharp absorption peak appearance at approximately ⿼228nm corresponded to a Ͽ⿿Ͽ* transition of the aromatic CC bonds [51].

Fig. 5.

UV⿿vis spectrum of GO aqueous solution.

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3.3.2Raman spectroscopy analysis

The Raman spectrum is a sensitive and informative technique to determine the disorders originated from sp2 carbon hexagonal networks with strong covalent bonds [52]. The Raman spectrum of GO was represented in Fig. 6. In this figure, two characteristic peaks of GO were clearly seen. The D peak appeared at 1358cm⿿1, and the G peak was detected at 1598cm⿿1. The prominent D peak is a sign of a reduction in the size of sp2 domains originated from the oxidation process, and the G peak is related with the graphitic order. Additionally, the intensity ratio of the D and G peaks (ID/IG) indicated that the number of defects due to the oxidation process [53]. The ratio, (ID/IG), was calculated as 1.01 for GO synthesized.

Fig. 6.

Raman spectrum of GO.

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3.3.3Transmission electron microscopy (TEM) analysis

The nanosheet morphology of GO was analyzed by TEM. From TEM images as presented in Fig. 7, the wrinkled layer morphology of GO could be clearly seen, which is in agreement with the literature [54].

Fig. 7.

TEM images of GO.

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3.4Characterization studies of scaffolds3.4.1Cell viability analysis

The MTT assay analysis was performed for evaluation of in-vitro cytotoxicity of produced scaffolds on MC3T3-E1 cells. The relative cell viability values were calculated using Eq. 2.2. The values were expressed as mean±relative standard deviation. The maximum relative standard deviation was calculated as 7.83% (Table 2).

Table 2.

Component ratio of scaffolds produced and their relative cell viability values.

Scaffold name  Scaffold component  Component ratio (wt%)Relative cell viability (%)  Relative standard deviation (%) 
Graphene Oxide1.0  30.58  4.95 
  Chitosan99.0     
Chitosan40.0  72.59  3.63 
  Hydroxyapatite60.0     
Graphene Oxide0.5  77.65  0.37 
  Chitosan39.5     
  Hydroxyapatite60.0     
Graphene Oxide1.0  159.26  2.51 
  Chitosan39.0     
  Hydroxyapatite60.0     
Graphene Oxide2.0  71.27  7.83 
  Chitosan38.0     
  Hydroxyapatite60.0     
FGraphene Oxide4.0  38.652.25
Chitosan36.0 
Hydroxyapatite60.0 

Two-component scaffolds, coded as scaffold A and B, were produced to compare with the three-component scaffolds, named as C, D, E, and F, in terms of their relative cell viabilities. The lowest cell proliferation was observed on scaffold A (Table 2), and this case could be explained by lack of 3D and porous structure of scaffold A. RCV% value of scaffold B was higher than scaffold A and slightly lower than scaffold C revealing that GO had proliferative effect on MC3T3-E1 cell line (Fig. 8).

Fig. 8.

MTT assay analysis of produced scaffolds.

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MTT assay analysis performed on three-component scaffolds fabricated, coded as scaffold C, D, E, and F, at various ratios of GO such as 0.5, 1, 2 and 4wt%, respectively, showed that scaffold D had the highest proliferative effect on MC3T3-E1 cell line. The proliferative effect was associated with the high osteoconductive effect of GO addition at a ratio of 1wt%. However, the increased GO ratio was inversely proportional with the MC3T3-E1 cell proliferation. This case could be explained by the dose-dependent toxicity of GO on the MC3T3-E1 cell line (Fig. 8).

As a conclusion, the scaffold coded as scaffold D, including 1wt% of GO, 39wt% of CS and 60wt% of HAp, had the highest relative cell viability value compared to the values obtained for other scaffolds produced. Therefore, the characterization studies were only performed for scaffold D by further FTIR, XRD, TGA, SEM, TEM, BET and mechanical compressive strength analyses.

3.4.2Fourier transform infrared spectroscopy (FTIR) analysis

The FTIR spectra of CS, GO, HAp and scaffold D were shown in Fig. 9. The stretching vibrations of CH at 2867 [42] and 1374cm⿿1, OCO at 1056 and 1027cm⿿1, NH2 at 3290 and 1418cm⿿1 were obtained in FTIR spectrum of chitosan [33]. The characteristic peaks of chitosan (Fig. 9) observed at 1652, 1587 and 1321cm⿿1 were attributed to amide I (CO) [42], amide II (NH) and amino (NH2) groups [33], respectively. The peaks observed at 1149 and 892cm⿿1 were attributed to ⿿C⿿O⿿C⿿ glycosidic bonding vibration of saccharides [42,55]. In the FTIR spectrum of GO (Fig. 9), the stretching vibration of OH was seen as a broad peak observed between 3000 and 3700cm⿿1. The characteristic peak of carboxyl CO stretching vibrations was seen in 1729cm⿿1. The peak corresponding to CC from unoxidized sp2 CC bonds was observed at 1619cm⿿1, and the peaks observed at 1044cm⿿1 are identified as COC vibrations [45]. For FTIR spectrum of HAp (Fig. 9), the sharp and strong band at 3573cm⿿1, and the peak observed at 628cm⿿1 corresponded to hydroxyl group (OH⿿) vibrations. The bands observed at 1088, 1026, 963, 599 and 566cm⿿1 were assigned to vibrations of the phosphate group, PO4⿿3[56].

Fig. 9.

FTIR spectra of CS, GO, HAp and scaffold D.

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In the FTIR spectrum of scaffold D (Fig. 9), all the characteristic peaks of individual components confirmed the dispersion of HAp and GO in CS. Additionally, the interactions among the components resulted in slightly shifted and reduced characteristic peak values. For instance, the intensity values of the peaks belong to ⿿NH vibrations of CS (at 1587cm⿿1) and CO stretch of carboxylic groups of GO (at 1729cm⿿1) significantly reduced due to the reaction of hydroxyl and amino groups presented in CS structure with carboxyl groups available in GO structure [38,43].

3.4.3X-ray powder diffraction (XRD) analysis

The XRD patterns of CS, GO, HAp and scaffold D were shown in Fig. 10. The characteristic peak of chitosan was observed at 2θ value of 19.91° and 26.63° (Fig. 10). The broadened peak around 2θ value of 22.23° represents the amorphous nature of CS [43]. In XRD spectrum of GO, the sharp characteristic peak of GO was seen at 2θ=9.85° (Fig. 10), and the peaks associated with graphite flakes could not be observed due to high ordered oxidation of graphite [53]. Two characteristic peaks of HAp were observed at 2θ value of 26.18° and 32.04° (Fig. 10) which are (002) and (211) reflections of HAp, respectively [44]. The size of the eggshell derived hydroxyapatite powder was calculated as 20.63nm by using Scherer formula as given in Eq. (2.1). In the XRD pattern of scaffold D presented in Fig. 10, the individual peaks belong to HAp was clearly observable unlike the individual peak belong to GO due to intense peaks at crystal planes of HAp and small content of GO. The strong and characteristic peaks of HAp suggested the stability of HAp in scaffold D. The presence of GO and CS were clearly confirmed by FTIR, SEM and TEM studies.

Fig. 10.

XRD patterns of CS, GO, HAp and scaffold D.

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3.4.4Thermogravimetric analysis (TGA)

Thermal degradation behavior of CS, GO, HAp and scaffold D was given in Fig. 11. The TGA profile of pure chitosan showed that thermal degradation of chitosan (Fig. 11) occurred in two stages. The first stage occurred in the range of 25⿿177°C due to the loss of water molecules. At the second stage, the primary degradation of chitosan started at 256°C, and it was completely degraded at approximately 815°C. According to the thermal behavior of GO given in Fig. 11, the relatively rapid weight loss of GO was observed below 100°C due to water evaporation. The destruction of functional groups containing oxygen atoms led to the second weight loss around 150°C. The final weight loss was observed around 230°C due to the carbon skeleton combustion of GO [33]. The HAp thermal behavior (Fig. 11) revealed that there is no significant change in the weight of HAp even at high-temperature values. This case indicated that HAp maintained its thermal stability. From the TGA profile of scaffold D (Fig. 11), the first weight loss was observed between 75 and 200°C due to the evaporation of absorbed water. The second weight loss was observed between 200 and 500°C due to thermal decomposition of chitosan and graphene oxide. At high temperatures (above 600°C), no significant weight loss was observed, and this case could be explained by HAp content (60wt%) of scaffold D.

Fig. 11.

TGA profiles of CS, GO, HAp and scaffold D.

(0.11MB).
3.4.5Scanning electron microscopy (SEM) analysis

Since the porous structure is an important parameter in terms of cell growth, the microstructure of scaffold D was analyzed by SEM (Fig. 12A⿿D). The porous structure of scaffold D and hydroxyapatite agglomerations were clearly seen in Figs. 12B and 12D, respectively. At lower magnifications, the wrinkled structure of GO was discernible. At higher magnifications, it was observed that GOCS was well bonded to the spherical HAp lattice. The curled and corrugated structure of GO enhanced the interlocking between GOCS sheets and HAp. Besides, the round-shaped macropores spread over the entire scaffold with pore sizes of 130⿿160μm were revealed at 100ÿ magnification (Fig. 13). The macropores are responsible for bone cell growth and nutrient delivery, while the nanopores are necessary for cell adhesion [57]. Thus, the porous sponge-like structure of scaffold with well-distributed HAp particles was supposed to facilitate cell growth and proliferation.

Fig. 12.

SEM images of scaffold D at 100 X, 263 X, 20.00 KX, and 50.00 KX magnifications.

(0.5MB).
Fig. 13.

Pore size of scaffold D at 100 X magnification.

(0.28MB).
3.4.6Transmission electron microscopy (TEM) analysis

In order to obtain further insight into the morphological structure of scaffold D, TEM analysis was performed. From TEM images represented in Fig. 14, HAp particles distributed on the transparent sheet-like structure which was attributed to the coating of chitosan layer on both sides of GO sheets. In these images, it was also clearly seen that HAp particles aggregated on some of GO-CS sheets due to their extremely high surface areas [35]. Additionally, the higher transparency of GO-CS sheets compared with GO sheets (Fig. 7) could be explained by the chemical interaction between GO and CS structures [58].

Fig. 14.

TEM images of scaffold D.

(0.24MB).
3.4.7Brunauer⿿Emmett⿿Teller (BET) analysis

For determination of specific surface area and pore size of scaffold D, Brunauer⿿Emmett⿿Teller (BET) analysis was performed. The N2 adsorption-desorption isotherm of scaffold D in Fig. 15 had a similar structure with that of Type IV isotherm with a H3 hysteresis [59]. This type of curve indicates the presence of the mesopores in the sample.

Fig. 15.

Nitrogen adsorption-desorption isotherm of scaffold D.

(0.1MB).

According to the parameters (presented in Table 3) calculated by Barrett⿿Joyner⿿Halenda (BJH) equation, scaffold D had a large BET surface area and total pore volume for cell attachment and proliferation [42,60]. The pore size distribution of scaffold D obtained by the BJH equation from the adsorption branch of the isotherm showed that the mesoporous structure was mainly distributed at 3.71nm (Fig. 16).

Table 3.

Surface area and porosity analysis of scaffold D.

Parameters  Value 
Surface area(BET) (m2/g)  15.345 
Pore volume(BJH adsorption) (cm3/g)  0.058 
Pore volume(BJH desorption) (cm3/g)  0.055 
Pore diameter(BJH adsorption) (nm)  3.710 
Pore diameter(BJH desorption) (nm)  3.403 
Average pore diameter (nm)  15.381 
Total pore volume (cm3/g)  0.059 
Fig. 16.

Pore size distribution of scaffold D.

(0.08MB).
3.4.8Mechanical analysis

The ideal scaffold should provide mechanical support for the cells to stimulate them for adhesion, proliferation, and differentiation. Scaffold B (0wt% GO) and scaffold D (1wt% GO) were analyzed to determine the effect of GO on the mechanical compressive strength (Fig. 17). The mechanical compressive strength values were expressed as mean±relative standard deviations. The maximum relative standard deviation was calculated as 1.35%. According to the data obtained, the mechanical compressive strength values of scaffold B and scaffold D were found as 128 and 244kPa, respectively. These results indicated that the mechanical compressive strength value of scaffold B (0wt% GO) almost doubled with scaffold D (1wt% GO). Even a small amount of GO addition into scaffold provided a significant improvement on its mechanical compressive strength. This result was also verified with the study performed by Rajesh and Ravichandran [21].

Fig. 17.

Mechanical compressive strength of scaffolds B and D.

(0.07MB).

In the meanwhile, the recent studies available in the literature, which performed on the fabrication of multi-component scaffolds for bone tissue engineering applications, were summarized on the basis of various specifications such as components used, crosslinking agents added, HAp synthesis method, drying method, mechanical compressive strength property, and cell viability ability, in Table 4. In these studies, the various components including natural polymers, inorganic materials, carbon-based nanomaterials, and proteins were incorporated to enhance the biological and/or mechanical properties of chitosan-hydroxyapatite (CS/HAp) scaffolds using the methods of freeze-drying, foaming, self-assembly, and sol-gel combined with 3D plotting. But, in the present study, the supercritical gel drying method was applied regarding its main advantage of complete solvent removal while keeping the macroporous structure of base materials of scaffold composite [61,62]. Besides, this method is an environmentally friendly process with mild-temperature, short processing time, an easy procedure providing easy control of the original morphology of scaffold [63], and enables the scaffold to have 3D porous structure without any physical collapsing in its structure [61,64] due to surface tension elimination [28,29,63,65]. But, in the case of conventional drying processes, the surface tension formation due to the vapor-liquid transition may cause a morphological collapse in scaffold structure during solvent removal. As a consequence, in the present study, three-dimensional scaffold, which fabricated by application of supercritical gel drying method, was found having the porous sponge-like structure with well-distributed HAp particles to facilitate cell growth and proliferation, and increased mechanical compressive strength, in comparison with other multi-component scaffolds fabricated at various structures/properties as represented in Table 4.

Table 4.

Recent studies on multi-component scaffolds for bone tissue engineering applications.

Reference  Components  Crosslinking agent  HAp synthesis method  Drying method  Mechanical compressive strength (kPa)  Cell viability 
Venkatesan et al. (2011) [30]Carbon nanotube  ⿿Derived from Thunnus Obesus boneFreeze drying⿿Increased cell proliferation
Chitosan 
HAp 
Venkatesan et al. (2012) [31]Chitosan  ⿿Derived from Thunnus Obesus boneFreeze drying⿿No toxic effect for osteoblast cells
Chondroitin sulphate 
HAp 
Venkatesan et al. (2012) [31]Chitosan  ⿿Derived from Thunnus Obesus boneFreeze drying⿿No toxic effect for osteoblast cells
Amylopectin 
HAp 
Pallela et al. (2012) [32]Collagen  ⿿Derived from Thunnus Obesus boneFreeze drying⿿High cell viability
Chitosan 
HAp 
Mohandes & Salavati-Niasari (2014) [33]GO  ⿿Precipitation method using Schiff baseFreeze drying⿿High bioactivity was observed in simulated body fluid
Chitosan 
HAp 
Shavandi et al. (2015) [34]Chitosan  Sodium tripolyphosphateDerived from waste mussel shellsFreeze dryingGood mechanical propertiesHigh cell proliferation
β-TCP 
HAp 
Kim et al. (2015) [35]Chitosan  0.2M CaCl2HydrothermalFreeze drying680kPa mechanical compressive strength for 70wt% HAp contentOsteoblastic differentiation was observed
Alginate 
HAp 
Sharma et al. (2016)[36]Chitosan  GlutaraldehydePurchasedFoamingMechanically stable scaffolds were producedHigh cell proliferation
Gelatin 
Alginate 
HAp 
Teimouri and Azadi (2017) [37]Chitosan  GlutaraldehydePrecipitationFreeze dryingGradually increased mechanical compressive strength values depending on HAp contentHigh cell attachment and high cell viability
Nanodiopsite 
HAp 
Yu et al. (2017) [38]Chitosan  GenipinPurchasedSelf-assembly processGood mechanical propertiesHigh cell proliferation
GO 
HAp 
El-Meliegy et al. (2018) [39]Dextran  ⿿PrecipitationFreeze dryingGradually increased mechanical compressive strength values depending on HAp content⿿
Chitosan 
HAp 
Dong et al. (2018) [40]Silica  ⿿Simultaneously synthesisSol-gel method combined with 3D plottingScaffolds having 10-13MPa mechanical compressive strength were producedHigh cell proliferation
Chitosan 
HAp 
Shahbazarab et al. (2018) [41]Zein  GlutaraldehydeHydrothermalFreeze dryingGradually increased mechanical compressive strength values depending on HAp contentHigh cell attachment and high cell viability
Chitosan 
HAp 
Türk et al. (2018) [42]Collagen  ⿿BiomineralizationFreeze dryingGood mechanical propertiesNo toxic effect for osteoblast cells
Multiwalled carbon nanotube 
Chitosan 
HAp 
Sumathra et al. (2018) [43]Graphene oxide  ⿿Simultaneously synthesis⿿⿿Encourage ability to cell propagation and cell expansion
Chitosan 
HAp 
Present studyGO  GlutaraldehydeDerived from eggshellsSupercritical gel dryingIncreased mechanical compressive strength value by addition of GOHigh cell proliferation
Chitosan 
HAp 
4Conclusions

In the present study, three⿿component scaffolds composed of GO, CS, and HAp by SC⿿CO2 assisted procedure were characterized in terms of their chemical, biological and mechanical compressive strength properties. The main findings were summarized as follows:

  • From MTT analysis, among other scaffolds fabricated, scaffold coded as D composed of GO (1wt%)/CS (39wt%)/HAp (60wt%), showed the highest cell viability ratio. GO additions into scaffolds above 1wt% ratio shown the dose-dependent cytotoxicity on MC3T3-E1 cells. Therefore, the characterization studies were performed only for scaffold D.

  • The peaks obtained from FTIR and XRD analysis for scaffold D confirmed that perfect blending of the components. The morphological structure analysis performed by SEM and TEM showed that scaffold D had a porous sponge-like structure, the macropores with pore sizes of 130⿿160μm, which is an essential property for its application in bone tissue materials.

  • From the BET analysis of scaffold D, surface area and total pore volume values were estimated as 15.345m2/g and 0.059cm3/g, respectively.

  • The mechanical compressive strength values of scaffolds coded as B with a GO ratio of 0.5wt% and D with a GO ratio of 1wt% were measured as 128 and 244kPa, respectively. This result revealed that GO addition was improved the mechanical compressive strength of scaffold. But, GO addition was limited due to its dose-dependent cytotoxicity even if it is an attractive agent to enhance the mechanical compressive strength of scaffold produced.

To sum up, the results of the original report showed that three-component scaffold coded as D, composed of graphene oxide synthesized by Improved Hummers Method (1wt%), chitosan purchased (39wt%) and hydroxyapatite derived from eggshells (60wt%), fabricated by application of the supercritical gel drying, had a three-dimensional porous sponge-like structure, highest relative cell viability, and increased mechanical compressive strength. The scaffold produced with these properties thought to be a promising candidate to be used for bone tissue materials. Besides, careful consideration on cytotoxicity, biocompatibility and structure-function relationship of the composites fabricated are required for their in vivo applications, which is part of the future work.

Acknowledgments

This work was supported by the Research Fund of Yildiz Technical University. Project Number: FBA-3210.

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