Research Article | Open Access
Aline Margarete Furuyama Lima, Marcelo de Freitas Lima, Odílio Benedito Garrido Assis, Alice Raabe, Hemelen Caroline Amoroso, Vera Aparecida de Oliveira Tiera, Marcelo Barbosa de Andrade, Marcio José Tiera, "Synthesis and Physicochemical Characterization of Multiwalled Carbon Nanotubes/Hydroxamic Alginate Nanocomposite Scaffolds", Journal of Nanomaterials, vol. 2018, Article ID 4218270, 12 pages, 2018. https://doi.org/10.1155/2018/4218270
Synthesis and Physicochemical Characterization of Multiwalled Carbon Nanotubes/Hydroxamic Alginate Nanocomposite Scaffolds
In this study, the preparation of porous nanocomposite scaffolds (HX-CNT) from a combination of a hydroxamic derivative of alginate (HX) and an amine-functionalized multiwalled carbon nanotube (CNT) at different concentrations is described. The structure of HX was investigated by FTIR, and the degree of substitution around 9% was confirmed by elemental analysis. The interaction between CNT and alginate derivative in the nanocomposite crosslinked with calcium was confirmed by FTIR, Raman spectroscopy, and SEM. The results obtained in this study showed that scaffolds based on HX-CNT composites with a 4 wt% concentration level exhibited improved physical and mechanical properties compared to plain alginate (Young’s modulus increased from 2.2 to 5.1 MPa and elastic strength from 0.13 to 0.25 MPa) and decreased the swelling ratios from ~900 to ~673. The cytotoxicity assays using the L929 cell line proved that the nanocomposite scaffolds were nontoxic, even at the highest CNT concentration.
Nanocomposite materials based on sodium alginate (SA) are used for different applications, such as support for enzyme immobilization , drug delivery carriers , electrochemical immunosensors , bioadsorbent of heavy metals  or ionic dyes , and biomaterials . To reinforce the alginate nanocomposites, various materials, usually natural fibres , hydroxyapatite , graphene oxide , or carbon nanotubes , are used to enhance the mechanical and physical properties for determined applications.
Alginate is a natural, anionic linear polysaccharide commercially obtained from brown algae. Chemically, the alginate is a linear block copolymer made up of 1,4-linked-β-D-mannuronic acid (M) and α-L-guluronic acid (G) units . In the form of sodium salt, alginate (SA) is soluble in aqueous neutral solutions, although it can become insoluble after its crosslinking [12, 13] with divalent cations such as Ca2+ forming a structure known as “egg box” . It is a biocompatible  and biodegradable  material, and also, the presence of carboxyl and hydroxyl groups on its structure allows modifications of its physicochemical and biological properties adapting the material for specific uses.
Among the strategies to obtain alginate derivatives, the chemical modification of polymers based on grafting with hydroxamic groups (RCONROH) has been shown to generate materials with important biological properties, such as in the inhibition of urease activity [15, 16] and as a potent inhibitor of matrix metalloproteinases . Similarly, pectin derivatives containing hydroxamic groups were reported to exhibit antioxidant  and significant inhibitory activities against semicarbazide-sensitive amine oxidase and the angiotensin-converting enzyme . Hydroxamic derivatives of alginic acid have also been reported to exhibit antioxidant properties  and potential for drug delivery systems .
Aiming to improve the physical and mechanical properties of alginate-based scaffolds, it was planned to merge the biological properties of its hydroxamic derivative with amine-functionalized multiwalled carbon nanotubes (CNT). The advantages of using carbon nanotubes are associated with their mechanical, thermal , and conductive  properties, making them suitable for nanocomposite preparation. Since their discovery in 1991 , carbon nanotubes have attracted a large amount of attention and have been indicated as an alternative additive to improve the mechanical properties of polymer matrices [25, 26]. A study  based on carboxylic-functionalized CNT alginate nanocomposite hydrogel prepared with different concentrations of carbon nanotubes (1, 3, and 5 mg/mL) was found to be able to improve the physical properties and showed that 1 mg/mL nanocomposite gel increased in stiffness and strength compared to pure alginate and the other nanocomposites. Besides, the nanocomposite hydrogels showed promising results by increasing the HeLa cell adhesion and proliferation. Other studies based on the nanocomposite of chitosan-decorated CNT/acrylamide-co-acrylic acid hydrogel  and graphene oxide/sodium alginate scaffold  showed improvement in the physical and mechanical properties dependent on CNT concentration.
In the present study, crosslinked nanocomposite scaffolds of a hydroxamic derivative of alginate were prepared by increasing the weight proportion of amine-functionalized CNT. The alginate derivative was characterized by FTIR and elemental analysis, and the nanocomposite scaffolds were prepared by a freeze-drying process and characterized by FTIR, mechanical strength, DSC, swelling ratio, Raman spectroscopy, and SEM. The results are discussed on the basis of the effect of the CNT content on the mechanical and physical properties of the formed scaffolds.
2. Experimental Part
Nanocomposite scaffolds were synthesized using sodium alginate from brown algae with viscosity higher than 2000 cP and viscosity average molecular weight of 475 kDa. Amine-functionalized multiwalled carbon nanotubes with 13–18 nm in diameter and purity > 99 wt% were purchased from http://CheapTubes.com (Cambridgeport, VT, USA). N,N-Dicyclohexylcarbodiimide (DCC), hydroxylamine hydrochloride, sodium hydroxide, acetone, ethanol, diethyl ether, calcium chloride, and hydrochloric acid were all purchased from Sigma-Aldrich Chemical Co., (St. Louis, USA). All solvents were reagent grade and were used as received. Spectra/pore membranes (Spectrum) were used for dialysis.
2.2. Synthesis of Hydroxamic Derivative of Sodium Alginate
Hydroxamic derivative of sodium alginate (HX) was prepared according to the literature  with 100% activation of carboxylic groups of sodium alginate using DCC. In this study, the derivative also underwent purification by dialysis (membrane of MWCO 12–14000 g·mol−1) against water for 7 days and was lyophilized.
2.3. Preparation of the Nanocomposite Scaffolds
In order to prepare the nanocomposite scaffolds with different properties, the CNT, in increasing amounts, were initially dispersed in aqueous solution of HX. The hydroxamic derivative solution (3 wt%) was prepared by dissolving 180 mg sodium alginate in 5 mL of sodium hydroxide solution under magnetic stirring overnight. After complete dissolution, the amine-functionalized CNT, previously dispersed in water (1 mL) using an ultrasonic bath for 1 h, was added, dropwise, to the derivative solution under stirring to obtain different mass percentages (0, 1, 2, 4, and 6 wt%) of CNT. The mixtures were maintained under magnetic stirring overnight to ensure a complete and homogeneous dispersion of the CNT. Scaffolds with only sodium alginate were prepared under identical conditions in water and at the same concentration as that used for the alginate derivative. The nanocomposite solutions were transferred to 96-well plates and cooled to −20°C in a freezer, and the frozen mixtures were lyophilized. The resulting scaffolds with different CNT proportions were designated as HX-1CNT, HX-2CNT, HX-4CNT, and HX-6CNT, where the number indicates the percentage of CNT added. The dry scaffolds were soaked in a 5 wt% CaCl2 solution for 12 h and then washed several times with water and again lyophilized.
The lyophilized HX and SA samples were characterized using an IRTracer-100 FTIR spectrophotometer (Shimadzu, Kyoto, Japan) using the KBr pellet method. The spectra were recorded from 4000 to 400 cm−1 at room temperature at a spectral resolution of 4 cm−1. An average of 32 scans was collected for each sample. The nanocomposite scaffolds were characterized by a VERTEX 70 ATR-FTIR spectrometer (Bruker, Billerica, USA).
2.4.2. Elemental Analysis
The sodium alginate and hydroxamic derivative alginate were investigated by elemental analysis using a 2400 CHN analyzer (PerkinElmer, Waltham, USA). Measurements were taken in duplicate.
The morphologies of the surfaces and cross-sections of the crosslinked nanocomposite scaffolds were examined using a scanning electron microscope (JSM 6510/JEOL). To observe the CNT in the scaffolds, the nanocomposites were observed under a JSM-6701F scanning electron microscope (JEOL, Akishima, Japan). Freeze-dried scaffolds were fixed on a carbon adhesive mounted over a SEM stub and coated with a thin layer of gold using a UNION Sputtering sputter coater (Oerlikon Balzers, Balzers, Liechtenstein). All the samples were examined under an accelerating voltage of 10.0 kV. The average pore size was estimated from the SEM images by using the ImageJ software (NIH, public domain).
2.4.4. Swelling Ratio (SR)
The SR of the scaffolds were quantified by rehydrating the dry samples with distilled water until equilibrium. Crosslinked nanocomposites, hydroxamic alginate, and sodium alginate scaffolds containing different dry weights were incubated in an aqueous medium at room temperature for 192 h. After removal, scaffolds were hung up until no dripping water was observed and were then weighed. The SR was expressed as a percentage of weight gain (water uptake) after immersion compared to initial dry weight, according to equation (1), where is the weight of the swollen scaffold and is the weight of the dry scaffold.
2.4.5. Compressive Mechanical Testing
The mechanical test of crosslinked alginate, hydroxamic alginate, and nanocomposite scaffolds was assessed by compression tests using a TA.XTPlus/50 texture analyser (Stable Micro Systems, Godalming, UK) fitted with a 5 kg load cell. Cylindrical scaffolds were compressed to 40% of their initial height at a compression speed of 0.01 mm·s−1. The stress-strain curve generated during the compression analysis was used to calculate the compressive Young’s modulus and elastic strength from the slope of the linear portion of the stress-strain curve. The data were expressed as (10).
2.4.6. DSC Measurements
Crosslinked composite scaffolds were subjected to DSC (DSC Q100/TA Instruments) to evaluate the transition temperature () to assess their thermal stability. was determined as the medium point of tangent lines traced at the beginning and at the levelling off of the DSC curve, corresponding to the inflection of the specific heat as a function of temperature. Samples (3 mg) were heated under dry nitrogen gas, and data were recorded using the following thermal cycle: a first cooling from room temperature to −70°C (at a rate of 30°C·min−1), equilibrating at −70°C, and then heating from −70°C to 140°C (at 5°C·min−1). After drying in a lyophilizer, all the scaffolds were kept in a desiccator. Prior to the measurements, the samples were maintained overnight at 50°C in a vacuum oven. An empty pan was used as reference.
2.4.7. Cell Viability
The indirect cytotoxicity test of the crosslinked scaffold was evaluated according to the literature  with some modifications. The L929 fibroblast cells were cultured with Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, and 1% penicillin-streptomycin and incubated at 37°C in a 5% CO2 atmosphere. Cells were seeded in triplicate in 96-well culture plates at a density of approximately in 200 μL of cell culture medium per well. The cells were cultured for 24 h at 37°C. The scaffolds were sterilised under UV light for 3 h and incubated at 37°C in a 5% CO2 atmosphere in triplicate in 2 mL of supplemented culture medium in 15 mL Falcon tubes. They were then incubated for 48 h to obtain the extract solution. The extract solution enriched in leachable released from scaffolds was diluted to 50%, 25%, and 12.5% in the same culture medium. After 24 h incubation, the extract (100 μL) was placed on the cell line and then incubated for a period of 24 h. The cell viability was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The absorbance was measured at 570 nm with an ELx808 universal microplate reader (BioTek Instruments, Winooski, USA).
2.4.8. Raman Spectroscopy
The HX-CNT composite samples with different CNT concentrations were characterized using a 633 nm line of a He-Ne laser on a LabRAM HR Evolution (HORIBA Scientific, France) Raman spectrometer at room temperature. The laser beam was focused on the samples by a microscope with a 50x objective, and the spectra were collected over the range of 800–2000 cm−1 and spectral resolution of 1.5 cm−1 and using a 600 lines/mm grating.
3. Results and Discussion
The hydroxamic derivative of sodium alginate, having the general structure of R-CO-NHOH, was synthesized by the nucleophilic attack of hydroxylamine at the carboxylic groups of the alginate by DCC, to obtain the HX derivative (Figure 1(a)). The sodium alginate was only partially modified; hence, the derivative still presents in its structure carboxylic groups in the acid form to enable electrostatic interactions with positively charged species.
The presence of hydroxamic groups in the alginate backbone was confirmed by FTIR and elemental analysis. The FTIR spectrum of the native sodium alginate (Figure 2(a)) exhibited bands at 3000–3600 cm−1 corresponding to O-H stretching and at 1613 cm−1 and 1415 cm−1, related to symmetrical and asymmetrical COO− stretching, respectively [31, 32]. The bands observed in the 1200–950 cm−1 range correspond to several typical vibrations of saccharide structures. A comparison between the spectrum of the sodium alginate and its derivative in Figure 2(b) revealed the band at 1638 cm−1, which corresponds to the hydroxamic groups . Furthermore, the spectrum of the modified alginate exhibited a band at 1741 cm−1 related to the carbonyl stretching mode of carboxylic acid .
The hydroxamic derivative of alginate was also characterized by elemental analysis, as shown in Table 1. The appearance of nitrogen in the analysis provides an additional confirmation of the insertion of the hydroxamic group onto the alginate backbone. The degree of substitution was estimated to be around 9%. Therefore, the FTIR spectra as well as the elementary analysis confirmed the success of obtaining the hydroxamic derivative from sodium alginate. The FTIR spectra from nanocomposite scaffolds with increasing proportions of CNT are shown in Figure 2(c). As can be seen from this figure, the addition of CNT reveals significant changes in the range of 400 to 2000 cm−1. As the amount of CNT increased in the HX nanocomposite, the intensity of the carbonyl stretching of carboxylic acid at 1741 cm−1 was gradually decreased.
Also, the FTIR spectrum for the nanocomposite scaffolds showed a shifting wavenumber from 1638 cm−1 to 1604 cm−1, which can result from interactions between the hydroxamic and carboxylic acid groups and the CNT (Figure 2(c)).
The further decrease in intensity of the abovementioned band and the observed band shifts may be credited to the electrostatic interactions between the amine groups of the functionalized CNT and the functional groups of the alginate derivative. In the literature [10, 33], the band shifting was also observed after introduction of carbon nanotubes when compared with the bands resulting from neat alginate. In another study , such decreased intensity has been attributed to the interaction of alginate with CNT and hydroxyapatite amide-functionalized carbon nanotubes.
The crosslinking with calcium was performed for sodium alginate and their hydroxamic derivative nanocomposite scaffolds. Calcium-crosslinked alginate is characterized by a reduction in the band intensity of asymmetric and symmetric stretching of COO−, along with a shift of the carbonyl band to a lower wavenumber from 1600 to 1590 cm−1 . Figures 2(d) and 2(e) show the FTIR spectrum of the HX-2CNT nanocomposite scaffold before and after the crosslinking with calcium. The crosslinking shifts the asymmetric peak of COO− stretching from 1601 cm−1 to 1596 cm−1; moreover, a shift from 1410 cm−1 to 1427 cm−1 corresponding to the COO− symmetric band was also observed. The asymmetric and symmetric stretching of COO− also becomes broader after crosslinking, suggesting an interaction of alginate with Ca2+ . Besides, the band assigned to carbonyl carboxylic groups at 1741 cm−1 vanishes or shifts to lower wavenumbers. The shift from 1738 cm−1 to 1637 cm−1 has been observed for iron-crosslinked hydroxamic alginate , and it has been attributed to complexation of carboxylic acid groups with iron ions. Hence, FTIR spectra demonstrated the calcium-crosslinked nanocomposites.
In this study, the amino-functionalized multiwalled carbon nanotubes were mixed with an alginate derivative solution to prepare the scaffold. Figure 3(a) shows that the carbon nanotubes were well dispersed throughout the alginate scaffolds and exhibit visually homogeneous scaffolds. The photo shows that the macroscopic appearance of the HX scaffolds presents a white colour and the gray colour turns darker as the amount of CNT increases from 1 wt% to 6 wt% mass. The SEM micrography in Figure 3(b) shows a low magnification of the dispersion of carbon nanotubes in the crosslinked HX-2CNT scaffold, and Figure 3(c), taken at a large magnification, confirms that the distribution of CNT in the scaffold is uniform in spite of the high concentration of CNT. This SEM micrography (Figure 3(c)) also shows that some CNT are embedded into the alginate scaffold, indicating a good interfacial interaction between polymer chains and carbon nanotubes, which was provided by the electrostatic interactions between the amino groups of CNT and the carboxylic groups. The porous structure of the crosslinked hydroxamic derivative of alginate and the nanocomposite scaffolds is visualized by SEM (Figure 4), after freeze drying and crosslinking.
The SEM micrographs of the crosslinked scaffolds showed interconnected pores in a random distribution of sizes. However, the average pore sizes increase after chemical derivatization, from 59 μm for the SA scaffold (Figure 4(b)) to 76 μm for the HX scaffold (Figure 4(a)). For the crosslinked nanocomposite scaffolds, the scaffold with 4 wt% of CNT (Figure 4(c)) resulted in a slightly rough surface when compared with the SA or HX scaffolds. Therefore, the presence of CNT may affect the formation of ice crystals on the surface of the scaffold during its preparation. In the present study, the addition of CNT increased its surface roughness (see Figure 4(c)) and induced a pore size reduction as the amount of CNT increased in the nanocomposite scaffold. The sizes of the pores estimated using the ImageJ software were , , , and for HX-1CNT, HX-2CNT, HX-4CNT, and HX-6CNT nanocomposite scaffolds, respectively. However, compared to the surface, the cross-section morphologies of the crosslinked nanocomposite scaffolds exhibited a highly interconnected porous structure, as shown in Figure 4(d). In the literature , the pore size of nanocomposite hydrogel increased as the concentration of CNT increased. The opposite effect was observed in graphene oxide/sodium alginate nanocomposites . In another study , the reducing pore size when the concentration of CNT is increasing may be due to increased viscosity of the nanocomposite solution.
Figure 5 shows the Raman spectra of HX-CNT composites with different CNT concentrations. The Raman spectra confirm the presence of CNT in the HX-CNT composites, with two characteristic bands at 1330 cm−1 (D-band) related to defective CNT and noncrystalline carbon  and 1593 cm−1 (G-band) that corresponds to the stretching of sp2 carbon atoms of graphitic materials .
The inset of Figure 5 shows the disorder of CNT using the intensity ratio between the D-band and the G-band . As the CNT concentration increased in the composites, the intensity ratio decreased. These results clearly confirm close intermolecular interactions between the CNT and the HX chains of the polysaccharide. In the literature , increasing the CNT concentration in the poly (glycerol sebacate) decreased the intensity ratio resulting from distortion of the CNT structure related to the interaction between nanoparticles and polymer chains.
The DSC tests for polysaccharides and the nanocomposite scaffolds were performed to evaluate the effect of both the chemical modification and the CNT percentage on the scaffold properties. Hence, in this work, refers to a process that comprises the water molecule content and the glass transition temperature. The curves are presented in Figures 6 and 7. In Figure 6, the of SA is found to be around 35.89°C and increases to 42.65°C, after calcium crosslinking network. The for crosslinking alginate scaffold is near to that previously reported by Lin and Yeh . The increase in can be attributed to the crosslinking process, which restricted the mobility of the polymer chains . The chemical modification of alginate with HX shifted the to 44.89°C (Figure 6(c)), and the calcium-crosslinked HX resulted in the largest change in the glass transition temperature, increasing from 44.89°C to 50.41°C (Figure 6(d)). Therefore, the results indicate that the insertion of hydroxamic groups strengthens the intra- and intermolecular hydrogen bonds and electrostatic interactions as well, consequently, decreasing the segmental mobility of the polymer chains and the amount of free water molecules.
The glass transition temperature (), of the crosslinked alginate derivative, and its calcium crosslinked nanocomposites are shown in Figure 7(a). Figure 7(b), obtained from the date of DSC curves, showed that the initially decreases for HX-1CNT and HX-2CNT nanocomposites and turns to increase as the percentage of CNT goes from 2% to 6%, indicating that the presence of CNT disturbs the crosslinking promoted by the Ca2+. It has been reported that, contrary to the classical egg-box model, Ca2+ ions may coordinate four carboxyl oxygens from carboxyl groups and four oxygen atoms belonging to water molecules . Hence, the hydrophobicity of CNT may disturb the “egg-box” and the water molecules involved with it, increasing the segmental mobility of the polymer chains. Other studies also reported the reduction in for carbon nanotubes/polycarbonate nanocomposites . As the percentage of CNT is increased, the presence of amino groups interferes with this effect by means of electrostatic interactions with the carboxyl groups and increases. Therefore, by varying the percentage of the CNT and the crosslinking with the calcium ion, scaffolds with varied properties can be obtained.
Figure 8 shows the swelling kinetics curves for the crosslinked alginate, the hydroxamic derivative, and its nanocomposite scaffolds. All scaffolds swelled rapidly at the start of the incubation () and, after one hour, no significant change was observed. The swelling of the crosslinked SA and HX scaffolds was very similar (~900%) to that recently obtained by Sarker et al. for calcium-crosslinked bioplotted scaffolds of SA . Interestingly, it is noteworthy that although the insertion of the hydroxamic groups have decreased the segmental mobility of the polymer chains into the HX scaffolds, the swelling ability was similar (Figure 8). However, the swelling behaviour of nanocomposites is markedly different from the pure polymers and it is closely related to the CNT percentage. The swelling ratios for the nanocomposites are slower than those for pure polymers and decrease as the proportion of CNT is increased. The HX-1CNT, HX-2CNT, HX-4CNT, and HX-6CNT nanocomposite scaffolds swelled by about 803%, 744%, 676%, and 673%, respectively.
Additionally, it is noticeable that the swelling ratio of the scaffolds became constant at around 72 h of immersion. According to SEM results, the surface of the crosslinked scaffolds of alginate becomes rough and the pore size decreases with increasing carbon nanotube concentration. This effect may be related to an increased physical crosslinking provided by the amino groups, hence, limiting the mobility of the chains . In addition, these interactions, as revealed by the infrared spectroscopy results, may decrease the hydrophilicity of the scaffolds and similar trends have been reported previously .
The compressive Young’s modulus and strength of the crosslinked scaffolds were measured, and the results are shown in Figure 9. The inset in Figure 9(a) shows the representative stress-strain curves of different scaffolds. It should be expected that scaffolds exhibiting a highly porous and interconnected structure, with varied pore sizes, could exhibit significant differences regarding their mechanical properties. From Figure 10, it can be seen that the compressive Young’s modulus is significantly affected by the hydroxamic groups as well as by the CNT content. As observed in DSC measurements, the presence of these groups increased the value of HX compared to SA (Figure 6). This improvement was also reflected in the mechanical properties, and hydroxamic groups almost doubled Young’s modulus compared to the alginate scaffold. Moreover, the moduli (Figure 9(a)) and elastic strength (Figure 9(b)) were shown to be highly dependent on the content of the CNT, i.e., when compared to the alginate derivative and Young’s modulus, and elastic strength for 2 wt% of CNT was twice those obtained for the HX scaffold. When the content of CNT was increased to 4 wt%, the elastic modulus and strength were enhanced from 2.2 to 5.1 MPa and from 0.13 to 0.25 MPa, respectively. Scaffolds of graphene oxide/sodium alginate showed improvement in elastic strength and Young’s modulus values compared to pure sodium alginate scaffold. In the literature, as the graphene oxide concentration increases to 5 wt% in graphene oxide/sodium alginate composites, Young’s modulus increases by 96.1% in comparison to the pure sodium alginate scaffold (from 1.205 to 2.333 MPa) . A similar improvement in Young’s modulus values was observed in acrylamide-co-acrylic acid nanocomposite hydrogel as increasing chitosan-decorated carbon nanotubes from 1 to 5% . In another study, nanocomposites of multiwalled carbon nanotubes and sodium alginate hydrogel showed that when the concentration of CNT increased from 1 to 3 mg/mL, Young’s modulus decreased and the explanation of this behaviour could be the weak chemical bond between CNT and the polymer structure .
In this study, it was confirmed that the homogeneous dispersion of the CNT in the scaffold, as observed in the SEM, and the interaction between CNT and the HX polymer as observed in the FTIR and Raman spectra resulted in an improvement in the mechanical properties. As estimated from SEM measurements, pore sizes decreased by about 20% as the percentage of CNT was increased to 6 wt%. It was evident that the interaction between CNT and HX chains synergistically played an important role in increasing the mechanical properties.
The cytotoxicity of the scaffolds was examined using L929 fibroblast-like cells, and the viability percentages were calculated in relation to cell control (Figure 10).
The results showed no cytotoxicity and the viability remained higher than 80%. It has been reported that nanocomposites from alginate and single-walled [46, 48] or multiwalled  carbon nanotubes showed nontoxicity to other cell types and improved cell adhesion and proliferation. It is clear that the crosslinked nanocomposites with 4 and 6 wt% of CNT show the best viability even at 100% extract concentration. In this study, amine-functionalized CNT has been incorporated in the nanocomposites, and as reported in previous studies [49, 50], the introduction of carbon derivative materials in extracellular matrix-derived substrate enhanced the cellular proliferation and adhesion. For alginate-graphene oxide matrices, it has been reported that the graphene oxide incorporation enhanced the cell viability . These results show that the nanocomposite scaffolds in this study are nontoxic and have a potential application as biomaterial.
The hydroxamic derivative of alginate was obtained and characterized by FTIR, and the degree of substitution around 9% was confirmed by elementary analysis. The HX-CNT nanocomposite scaffold was prepared by a freeze-drying technique. The interactions between CNT and alginate were confirmed by FTIR, DSC, Raman spectroscopy, and SEM results. The compressive Young’s modulus and elastic strength of the nanocomposite scaffolds were found to be above those of the pure alginate and hydroxamic derivative scaffolds. All the scaffolds showed interconnected pores, and CNT affected the scaffold formation by decreasing the pore size and swelling ratio and increasing Young’s modulus as the CNT quantity was increased in the nanocomposites. The cytotoxicity assay using the L929 cell line showed that nanocomposite scaffolds are nontoxic, even at higher CNT concentrations. The results from this study concluded that hydroxamic alginate and CNT nanocomposite scaffolds exhibit improvement in the physical and mechanical properties and can be further explored for in vivo applications.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001. Aline M. F. Lima is thankful to Dr. Ana Carolina Conti e Silva and Alana Lisbôa da Silveira from the Departamento de Engenharia e Tecnologia de Alimentos for their assistance with the mechanical property measurements. M. B. Andrade thanks Conselho Nacional de Desenvolvimento Científico e Tecnológico for grant 113844/2018-2. The authors are also thankful to Glaura Goulart Silva from the Federal University of Minas Gerais for supplying CNT and RedeAgroNano (Embrapa) and MCTI/SisNANO for the financial support.
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