Cellulose
Cellulose occurs in almost pure form in cotton fibers; however, in plants, it is found in conjugation with other raw materials, such as lignin and hemicelluloses (Kalia et al. 2011; Meng et al. 2019). In the current study, cellulose was therefore isolated from BPS using the liquefaction method followed by the bleaching process. In this strategy, impurities, including lignin and hemicelluloses can be solubilized by liquefaction, while the residual lignin can be removed using the bleaching process (Meng et al. 2019). The resulting BLR was then used as the polymeric material for fabrication of cellulose/HA nanofibers. The overall macroscopic appearance for the BPS after two steps of treatment is shown in Fig. 1a. According to the figure, the milled light brown BPS powder changed to brownish-black residue (i.e., LR) as a result of the creation of some chromophore during the liquefaction treatment. However, the bleached component (i.e., BLR) appears as a white powder, confirming that hemicellulose, lignin and other non-cellulosic substances were completely removed by the extraction process; accordingly, the final product is suggested to be almost pure cellulosic material. Fig. 1b shows FTIR spectra of BPS, LR and BLR to compare the change in chemical structure resulting from this purification process. All spectra exhibited the typical characteristic bands of cellulose, e.g. 3100–3500 cm−1 (OH stretching), 2900 cm−1 (CH stretching), 1450 cm−1 (CH2 bending), 1305 cm−1 (CH asymmetric deformations) and 1035–1095 cm−1 (CO stretching) (Li et al. 2015; Meng et al. 2019). In contrast, no significant absorption bands at 1735 and 1244 cm−1 due to C=O stretching vibrations of lignin and hemicellulose and C-O-C vibrations of aromatic ether linkages in lignin can be detected in BLR spectrum, indicating that lignin and hemicellulose content can be relatively removed after the liquefaction and bleaching processes. Therefore, the results indicated that the chemical structure of cellulose chains remains unchanged following the conducted treatments.
To examine the crystalline structure of the produced cellulose, XRD analysis was used as depicted in Fig. 1c. Indeed, its linearity along with hydrogen bonding interactions and van der Waals forces between adjacent molecules can induce a closely packed crystalline structure in cellulose (Zhang et al. 2004). According to the diffraction pattern of BLR, two peaks at around 2θ = 22° and 15.3°, corresponding to the 2 0 0 and 1 1 0 plane positions, can be detected confirming a typical crystalline polymorph of cellulose I (French 2014; Li et al. 2015; Meng et al. 2019). The Cr.I. and thickness of cellulose crystallites determined respectively using Eqs. 1 and 2 were 51.8% and 5.22 nm respectively, which were very close to the values reported in the literature (Manimaran et al. 2020; Pelissari et al. 2014). While, the Cr.I. for naturally produced BPS has been reported around 8.7%. (Meng et al. 2019), the higher degree of crystallinity of BLR compared to BPS can be attributed to the progressive removal of amorphous hemicellulose, lignin and other non-cellulosic contents due to the successive liquefaction and bleaching treatments. To study whether the producing cellulose was suitable for the electrospinning, we also assessed the MW of BLR with the well-known Mark-Houwink equation which relates the MW of cellulose to [Ƞ] (Eq. 3). While, many characteristics of cellulose depend on its chain length or the degree of polymerization, MW of the extracted cellulose from various plant sources are reported between 1.0 × 104 and 8.0 × 105 (Immergut et al. 2953). In a typical electrospinning process, higher molecular weights of the polymer (i.e., larger chains) favors uniform fiber formation. On the other hand, number of beads in the fiber structure decreases with increasing MW (Gupta et al. 2005; Rezaei et al. 2015). According to our viscometry and Eq. 3, the estimated MW of the produced cellulose was around 3.0 × 105 which is acceptable for a plant-origin cellulosic polymer.
HA nanoparticles
Nanosized HA particles have long been applied as reinforcing agent in polymeric nanocomposites, as they can significantly enhance their biocompatibility, bioactivity, osteoconductivity and mechanical strength (Sadat-Shojai 2015). In the current study, the as-synthesized HA nanoparticles were characterized in terms of their morphology, chemical structure, and bioactivity. The colloidal stability of HA in BLR solution was also investigated. According to XRD analysis (Fig. 2b) and the ICDD standard card no. 00-09-0432, the as-synthesized powder was relatively pure HA phase with a hexagonal crystal system. From the XRD data, the crystallinity index and the crystallite size (for isolated peak assigned to 2 0 0 plane) was 0.80 and 43 nm, respectively. Moreover, the IR spectrum depicted in Fig. 2b confirmed the chemical composition of the precipitate in accordance with the formula of HA (i.e., Ca10(PO4)6(OH)2) (Pang and Bao 2003; Sadat-Shojai and Moghaddas 2020). Fig. 2c shows SEM and TEM micrographs of the powder, indicating that prepared particles were relatively irregular in shape with an average grain size less than 70 nm. Since the purpose of this study was to fabricate bone scaffolds, it was necessary to examine the in vitro bioactive properties of the as-synthesized particles. Fig. 2d shows the SEM of particles after 30 days immersion in SBF at 37°C, indicating generation of a flake-like microstructure as a result of nucleation of new minerals on the surface of nanoparticles. Accordingly, the HA powder prepared here have relatively high bioactivity and hence may be a good candidate as reinforcing agent in polymer scaffolds. A colloidal stability study which is usually considered as the sedimentation behavior of particles was performed to investigate the duration of HA dispersion in BLR/TFA solution. According to the results (Fig. 3), there is not any significant aggregation and/or precipitation of nanoparticles until 1 h after the sonication. Indeed, during one hour of monitoring, the suspension containing HA nanoparticles remained completely turbid. As the electrospinning of cellulosic nanofibers containing particles takes a maximum of 1 h, these results can guarantee that nanoparticles can remain stable in the organic phase during the process. However, as indicated in Fig. 3, for longer times (up to 6 hours) some nanoparticles begin to precipitate and some others dissolve in the acidic solution, indicating that the as-synthesized powder cannot remain stable for long periods.
Analysis of the electrospinning experiments
The DOE is known to be a powerful tool for achieving specific goals, simultaneously examining several control factors, and systematically evaluating empirical data (Gadomska-Gajadhur et al. 2018; Smiciklas et al. 2005). Among the possible DOE methods, Taguchi design, as an orthogonal array method, can optimize several processing variables and extract large quantitative information from just a few experimental trials. In addition, this method efficiently identifies those input variables which have the major impact on the selected response (Sadat-Shojai and Ghadiri-Ghalenazeri 2020). Therefore, in the fabrication of cellulosic nanofibers using electrospinning method, a Taguchi experimental design based on an L9 orthogonal array seems to be an appropriate approach to optimize the processing variables and to study their influence on the fiber diameter and degree of splashing. For this, among the factors that may influence the electrospun fibers, solution concentration, voltage, flow rate and WD were evaluated using a three-level Taguchi series experimental design approach. In this strategy, the response of the fiber diameter and degree of splashing were determined by image analysis of SEM micrographs.
It is well-known that critical characteristics of electrospun fibers, such as mechanical strength and toxicity to cells depend strongly upon their microstructure, mainly their morphology, orientation, uniformity, and fiber diameter (Bombin et al. 2020; Hooshmand-Ardakani et al. 2020; Sadat-Shojai and Moghaddas 2020; Sadat-Shojai and Moghaddas 2021; Zarei et al. 2021). However, when one considers the nanofibers, their dimensions seemed to be highlighted more than other characteristics. Figs. 4 and 5a show SEM photomicrographs and diagram of estimated dimensions of the electrospun nanofibers, respectively. According to the SEM observations, two types of morphologies were formed depending on the processing conditions. As seen in SEM images of run no. 1 to run no. 5, a non-uniform fibrous structure with a large amount of splashing was obtained through electrospinning at low solution concentrations (i.e., 1 and 1.4 wt.%). However, Fig. 4 shows, while an increase in solution concentrations to 1.6 wt.% with a higher applied voltage of 25 kV (i.e., run no. 8) resulted in a uniform structure, a further increase in voltage to 30 kV with the minimum working distance of 10 cm (i.e., run no. 9) yielded a very uniform and interesting nanofibrous mat. According to Fig. 5a, among the studied variables, the high influence of the solution concentration is evident by the significant increase in fiber diameter with increasing the solution concentration. Since fibers are generated by evaporation of the solution jet, the diameter of the nanofibers largely depends on the size of the jet as well as the concentration of polymer in the solution jet. Several researchers have suggested that the higher the viscosity of the electrospinning solution, the larger the diameter of the generated fibers (Nezarati et al. 2013; Zeng et al. 2003). Accordingly, the reason behind the increase in the fiber diameter, as shown, is the increase in solution viscosity and the difficulty of pulling the jet at higher concentrations. Moreover, at higher cellulose concentrations, the amount of solvent in the solution jet is less and hence the solvent can evaporate faster. Therefore, with a more concentrated solution, drawing the jet becomes more difficult and dry fibers with larger diameters are generated on the collector. Although smaller diameters for nanofibers may be preferred due to their higher similarity to the natural ECM, as shown in Fig. 5b, the amount of solution splashing (i.e., droplet formation) decreases significantly with increasing fiber diameter from run no. 1 to run no. 9. Indeed, for low-viscosity solution the polymer chains are not entangled and jet is unstable and breaks up into droplets; accordingly to achieve fibers with a lower splashing, solutions with higher concentrations should be used.
A graphical display of the level effect of each electrospinning variable is given in the mean plots in Fig. 5c, in which analyses of the magnitude and the importance of each input factor’s effect are shown. In addition, Table 2 indicates the maximum S/N ratios generated at the optimum level. Again, according to Fig. 5c and Table 2, the cellulose concentration of the electrospinning solution has the greatest effect on the selected responses. Moreover, while the applied voltage during the electrospinning process can also affect the fiber diameter and amount of splashing, based on the S/N ratios, the flow rate and working distance have the lowest importance among the studied factors. From these results, the optimum level for each electrospinning parameter was confirmed as: solution concentration of 1.6 wt.%; applied voltage of 30 kV; flow rate of 0.5 mL/h and WD of 10 cm.
Cellulose/HA nanofibers
Although electrospun nanofibers from native cellulose have been previously reported, very few studies, to the best of our knowledge, were focused on development of electrospun cellulose nanofibers containing HA nanoparticles. Moreover, a comprehensive characterization of cellulose nanofibers have rarely been reported before. However, it is expected that stiff and bioactive HA nanoparticles would remarkably influence the structural, mechanical and biological properties of the electrospun cellulosic mats. The morphological characteristics of the as-spun composite fibers were examined using SEM as shown in Fig. 6a. As can be seen, due to the optimization of the electrospinning conditions, the created nanofibrous structures are uniform with minimal bead and splashing defects. Indeed, the absence of beads and splashing droplets can increase surface area-to-volume ratio of the fibers, which in turn increases the contact area between the fibers and the living cells. On the other hand, according to SEM observations, there is not any significant particle agglomeration and/or aggregation in C-HA10 and C-HA20 samples, suggesting that HA nanoparticles could be homogeneously embedded in the cellulose nanofibers at the expected concentrations. The complete encapsulation of nanoparticles may result from significantly larger diameter of the nanofibers compared with that of the nanoparticles. To further confirm the homogeneous distribution and dispersion of filler inside the fibrous matrix, EDX-mapping of nanoparticles for C-HA20 sample was also carried out as shown in Fig. 6b. Again, the results clearly suggested that HA nanoparticles were well encapsulated and evenly dispersed inside the whole fibrous mat.
Diameter of the as-spun fibers were determined from SEM images and the results were summarized in Fig. 6c. According to the results, the diameters of the nanofibers decreased non-significantly with the addition of nanoparticles. The observed decrease in the fiber diameter with increasing concentration of filler may result from the fact that reduction of viscosity of the solution jet near the collector can occur due to the lower fraction of the polymer. Interconnected porosity is another essential requirement for artificial tissue engineering scaffolds. Indeed, scaffolds should mimic the native ECM in terms of porosity to allow proper cell and nutrient migration (Bombin et al. 2020; Sadat-Shojai and Arvaneh 2021; Zarei et al. 2021). The porosity degrees calculated using Eq. 5 were summarized in Fig. 6d. According to the figure, there was a non-significant increase from 61–69% in porosity with increasing HA concentration, suggesting that the degree of porosity may slightly be controlled with the filler concentration, although more investigations is needed to determine the reason behind this effect.
An important characteristic to be considered for bone scaffolds is mechanical strength. Generally, the mechanical strength of an electrospun fibrous scaffold is controlled by several parameters, including intrinsic characteristics of the polymer (mainly its molecular weight), degree of porosity, average diameter size of fibers, and presence of defects (Han et al. 2020; Tarus et al. 2016). Fig. 7a illustrates the representative stress-strain curves of C-HA0, C-HA10 and C-HA20 fibrous samples. From these curves and their extracted data, the elastic moduli and the tensile strengths of the samples were determined as shown in Figs. 7b and 7c respectively. According to the results (Fig. 7b), the samples containing HA have a higher elastic modulus with respect to the sample C-HA0, owing to the incorporation of HA as a stiff filler. Moreover, the amount of modulus increased significantly with increasing the filler concentration. The obtained results were in agreement with the results of our previous studies in which mechanical strength of various polymers was a function of the filler content (Sadat-Shojai 2015; Sadat-Shojai and Moghaddas 2020; Sadat-Shojai and Moghaddas 2021). In fact, when the high modulus HA reinforcement was conjugated with the lower modulus cellulose, applied stress transfers from the polymeric matrix to the stiff filler, leading to an increase in the average modulus. The addition of high-modulus HA increased the tensile strength of the cellulose as well. As shown in Fig. 7c, sample C-HA20 has the highest tensile strength, which indicates a direct relationship between the addition of HA and tensile strength.
Fabricating electrospun mat with satisfactory biological properties is one of the great challenges in attempting to design bone scaffolds. In the current study, to investigate the biological properties of the fabricated nanocomposites, their degree of wetting was first determined by using contact angle measurement. Contact angle, in turns, depends on several parameters including hydrophobicity of nanocomposite, surface roughness and degree of porosity (Gu et al. 2016; Sadat-Shojai and Moghaddas 2020). According to the results (Fig. 8a), the nanocomposite containing the highest amount of filler (i.e., C-HA20) exhibited a water contact angle of 91 ± 5.7 °, which was significantly smaller than control (C-HA0, 112 ± 5.6 °). The greater wettability of C-HA20 should obviously be attributed to the incorporation of HA particles as a hydrophilic filler. Biodegradability analysis of biomaterials provides a useful tool to assay their suitability for tissue engineering applications. Indeed, scaffolds should be degradable naturally over the time to permit new tissue to form. The weight loss of the C-HA20 sample with regard to degradation time is shown in Fig. 8b, suggesting that around 5% weight loss can occur after six weeks incubation in PBS at 37°C. It is noticeable that two other samples including C-HA10 and C-HA0 did not show a significant weight loss until the sixth week in vitro, indicating that the prepared cellulose is hardly degradable in aqueous solutions with neutral pH and the absence of enzymes. On the other hand, the relative biodegradability of C-HA20 was due to its high concentration of incorporated HA, which can increase the water uptake and hence adjust its degradation rate. The bioactivity of fibrous filled nanocomposite with the highest HA concentration (i.e., C-HA20) was evaluated after soaking in SBF solution for 30 days under physiological conditions. Fig. 8c shows an interesting apatite particles aggregates forming a flake-like structure deposit on the surface of the nanofibers. This is surprising because according to the morphological studies (Fig. 6a), almost all HA nanoparticles were encapsulated inside the fibers and therefore there was no direct contact between the nanoparticles and the SBF liquid. Overall, these results confirmed that the fabricated scaffold has bioactivity and has the ability to form a new apatite phase on its surfaces, which in turn leads to a direct bonding to the living bone after its implantation in the body.