3.1 RSM statistical study (The effect of SA:PVA ratio and The effect of the operating parameters)
In this research, it was tried to improve the electrospinning potential of SA by combination with PVA and also study the electrospinning main parameters (V, Q, and X) to produce nanofibers with better quality. To aim this, As can be seen in Table 1, 13 runs have been considered according to the RSM study to find out the nanofiber producibility of each formulation of SA and PVA. Table 2, represents 20 different conditions to produce nanofibers. The DOE software provided quadratic equations as the governing relations between the percentage of ingredients and the selected response (nanofiber production) were examined via ANOVA. Table 3 summarizes the results.
The reliability of a model is usually justified via P-value, which should be lower than 0.05 to conclude that the model fitting the experimental data is valid and significant [31]. As can be seen in Table 1, the P-value was lower than 0.05 in both studies. Considering the effect of the percentage of the SA and PVA on nanofiber producibility, P-value was higher than 0.05 for A and B (as the first-order effects ), AB (interaction effect) and B2 (as the second-order effects). P-value was lower for A2 as the second-order effect of PVA (Fig.1). Regarding the effect of operation parameters, P-value has been reported lower than 0.05 which depicts the validity and significance of the governed equation. The P-value was lower than 0.05 only for A as the first-order effect, AB as the interaction order, C2 as the second-order effect. P-value was too high for AC and BC and C.
Table 3: The governed equations and the relevant analysis of variance results
Response
|
Final equation in terms of code factors
|
P-value
|
R2
|
Adj. R2
|
AP
|
Nanofiber producibility*
|
2.97 - 0.17 A - 0.17 B - 0.25 AB - 1.88 A2 + 0.12 B2
|
0.01
|
0.83
|
0.72
|
6.83
|
Nanofiber producibility**
|
3.4 + 0.9A + 0.5B -0.1 C + 0.75 AB – 0.5 A2 + 0.5 B2 – 1.5 C2
|
0.016
|
0.79
|
0.61
|
6.3
|
* A: PVA – B: SA , ** A:V , B: X, C: Q
The reliability of a fitted model is specified by the determinant coefficient (R2) and Adj.R2 as its adjusted form. The validity of the model can be approved if R2 ≥ 0.6 [32]. both models showed R2 equal 0.83 and 0.79 and had a reasonable agreement with adj.R2 indicating that the models are capable to analyze and predict the response over the change in the process parameters. Adequate precision (AP) compares the range of the predicted values at the design points to the average prediction error, where a ratio higher than 4 is desirable [33]. As Table 3 depicts, in both models, AP values reported higher than 4, showing that there was a good agreement between the predicted and experimental values including most of the responses.
Fig.1 shows the relation of response with the effect of the SA and PVA combination. Based on the contour results, a higher number of their combination fail in nanofiber production and the likelihood of nanofiber producibility was too low. According to the experimental results, the only SA:PVA ratio that showed spinnability was 1:6.5. In other cases, no nanofiber was produced due to high or low viscosity and lack of enough surface tension. SA did not show spinnability when employed lonely. The reason might be related to the limited solubility and high viscosity of this natural polyelectrolytic polymer. Previous studies reported that the combination of SA with other polymers increases the spinnability of SA [34, 35]. Due to the formed hydrogen bonds between the SA and other polymers such as PCL, the repulsive force between the polyionic molecules is notably reduced to boost chain fusion, which finally leads to the nanofibers production [36]. For instance, Gong and his colleagues produced SA-based nanofibers by employing polyethylene oxide (PEO) [33]. Lu et al. [37] studied the electrospinning ability of SA in combination with PEO at a concentration of 1 to 4%. They showed that only 3% of PEO resulted in smooth and uniform nanofibers. It was reported that the final viscosity plays a vital role in spinnability [38]. In some cases, addition of Surfactants such as triton-X100 can improve the viscosity and also the spinnability [39]. Based on the obtained governed equation in Table 3, it seems that adition of PVA in each concentration did not guarantee the spinnability of SA and only the 6.5% of PVA combined with 1 % of SA resulted in nanofibers. The mentioned ratio was considered as the optimized SA:PVA ratio. To analyze the nanofiber and also the effect of process parameters on the quality of the synthesized nanofibers, this ratio was used as the main formulation for the rest of the study. Other ratios did not result in nanofiber production under any adjustment of operating parameters including voltage (0-30 kV), the working distance (5-20 cm), and flow rate (0.1-1 µl/h).
The effect of X and V: As can be seen in Fig.2-A, at constant Q, low V affects the nanofiber production negatively. To produce nanofibers at low V, it is necessary to decrease the working distance (lower X). However nanofiber production at a higher voltage and working distance is more possible compared with lower levels.
The effect of V and Q: At constant X, Fig. 2-B shows that the likelihood of nanofiber production can be increased by optimization of the Q. Fig.2-B also depicts that by enhancing V, the Q must be adjusted at an average flow rate which means in higher or lower Q (set in range) spinnability decreases. However, the distance needs to be adjusted (Fig.2-A).
The effect of Q and X: Fig.2-C illustrates that adjusting Q in a high or low rate (at constant V) cannot result in nanofiber production possibility. The appropriate Q seemed to be set around 0.5 ml/h but in high working distances. Under this condition, spinnability is more improved.
In general, V, Q, and X need to be adjusted to increase spinnability. Based on the results the central points for V, Q, and X seemed to be the apropriate levels. The applied voltage is a critical factor in electrospinning for spinnability. This is because nanofiber production only happens when the applied voltage exceeds the threshold voltage [40]. In the case of voltage, levels equal or above 15.5 kV showed better improvement. In similar research, the voltage between 12.5-24 kV was reported as appropriate V for nanofiber production of SA/PEO. It was reported that too high or too low V fails spinnability [41]. According to the previous reports, increasing the applied voltage increases the electrostatic force of the polymer solution, which is visible in jet traction, and ultimately reduces the length of the nanofibers [42]. It has also been reported that changing the applied voltage changes the quality of the nanofibers, thereby changes the diameter and morphology of the nanofibers [43]. Reneker et al. [44] stated that enhancement in the applied voltage has no effect on the fiber diameter of PEO. However, in 2005 Zhang and his colleagues reported obtaining larger-diameter nanofibers higher voltages must be applied because it causes more polymer ejection [45]. Interestingly, Other scientists have reported that an increase in the applied voltage decreases the nanofiber diameter. Furthermore, it was observed that at higher applied voltages, bead formation was obtained on the fibers [42, 46].
Another parameter that affects the control of morphology and diameter of nanofibers is the distance of the nozzle from the collector. To prevent evaporation of the polymer solution before the fiber reaches the collector, it is necessary to optimize the distance [47]. Therefore, in the electrospinning method, an optimized distance is required to fibers reach the collector and prevent solvent evaporation. Based on the results of our study, this distance depends entirely on the applied voltage and the flow rate. Longer distances have been reported to produce thinner fibers [48] but this claim is true when increasing the distance does not have a negative effect on fiber formation and power outages [49]. Also, beads will appear when they are too close or too far [43, 50]. In a study, it was reported that increasing the working distance caused an increase in diameter [51]. Interestingly, because the fibers need to be cooled to achieve uniform fibers and to prevent fiber fusion, shorter collector distances can increase the likelihood of fiber fusion at the junction.
Polymer flow rate indicates the flow rate of the polymer solution per unit time, which is known as another factor affecting the quality of fibers. It has been reported that increasing the flow rate causes more polymeric material to come out of the nozzle, which leads to the production of coarser fibers. Low flow rates are essential for the production of good quality fibers with uniform diameters [52]. It has been predicted that nanofiber diameter decreases due to increased charge density at low rates [42]. It was also reported that with increasing flow rate, there is a continuous increase in nanofiber diameter [44]. It is notable that an excessive increase in flow rate not only enhances the integration of nanofibers but also creates a bead in the fiber structure because not enough time is provided for solvent evaporation.
Experimental results showed that only the following runs were succeeded in electrospinning: 2-3-5-7-8-11-12-14-15-16-17-18-20. Amongst them, run numbers of 7-8-11-16-17-20 were considered as the repeated runs to evaluate the validity of the experiment and monitoring the errors from the operator. The appearance evaluation and also the SEM analysis of these groups (data not provided) were the same and run 8 was considered as the representative of these runs. Hence, the only groups employed in the next analysis were 3-5-7-8-12-14-15-18. In the rest, they are named Scaffold 3, Scaffold 5, …, Scaffold 18.
3.2 Morphology and physical evaluation of the nominated scaffolds.
Fig.3 and Fig.4 show the SEM images of the scaffolds with two magnifications and fiber diameter distribution. The porosity and the fiber diameters are also reported in Table 4. As can be seen, scaffolds depicted differences in nanofiber density, uniform distribution, nanofiber diameter, and the quality of electrospinning with fewer or no beads. Fig.3 shows that scaffolds 3 and 12 did not have uniform nanofibers in size distribution and quality. The voltage was equal for both scaffolds but they were different in distance and flow rate. The scaffolds 2, 5, and 7 although had uniform nanofibers but showed a low density of nanofibers The reason can be attributed to the disproportion of flow rate to distance. Low flow rate (< 0.55 µl/h) towards the voltage ( >15 kV) can be considered as the main reason for low density. This is while the scaffolds 14, 15, and 18 illustrated the best results in density, uniform nanofibers and smooth fibers. According to Table 4, the lowest and highest porosity belonged the scaffold 14 (521 nm2) and 3 (1404 nm2) respectively. Scaffolds 2 and 7 also had high porosity equal to 1004 and 1205 nm2 respectively. Considering the size distribution of nanofiber, Table 4 also shows that the thin nanofiber belonged to the scaffold 12 and 15 (140-170 nm) while the scaffold 3 and 7 owned the thick nanofibers (300±5 nm respectively). Scaffold s 2, 5, 14, and 18 showed nanofibers in the range of 220-240 nm. It can be hypothesized that applying higher voltage between 15.5-30 kV, adjusting the distance between 12.5-20 cm, and providing the flow rate at 0.5-1 µl/h resulted in appropriate nanofibers. The results were in agreement with previous studies. Hu and his colleagues produced SA/PEO nanofibers with 120-160 nm in diameters under 12, 18 and 24 kV [41]. The little difference may be attributed to the process parameters. In another study, the SA/PVA nanofibers were produced with 140-350 nm diameter [53]. Table 4 also depicts the results from the contact angle analysis which due to the high hydrophilic feature of both SA and PVA, the reported contact angle for all scaffolds was lower than 5ᵒ meaning that the scaffolds are extremely hydrophlilic.
Table 4: The physical properties of the elected scaffolds.
Scaffold
|
Porosity Area (nm2)
|
Fiber mean diameter (nm)
|
Contact angle
|
scaffold 2
|
1004.668
|
222.6
|
< 5 ᵒ
|
scaffold 3
|
1404.154
|
296.5
|
< 5 ᵒ
|
scaffold 5
|
791.831
|
239.5
|
< 5 ᵒ
|
scaffold 7
|
1205.018
|
307
|
< 5 ᵒ
|
scaffold 12
|
556.001
|
137
|
< 5 ᵒ
|
scaffold 14
|
521.317
|
210
|
< 5 ᵒ
|
scaffold 15
|
619.965
|
166
|
< 5 ᵒ
|
scaffold 18
|
829.567
|
227
|
< 5 ᵒ
|
According to the results from the morphology analysis and quality evaluation of the synthesized nanofibers, Scaffold 14, 15 and 18 were nominated for more analysis. These scaffolds revealed appropriate density, smooth nanofibers, uniform size distribution and suitable porosity. In the rest of the study, the scaffolds were first crosslinked under 25% glutaraldehyde vapor and then were evaluated.
3.3 chemical structure
Fig.5 shows the FTIR spectra of the three nominated crosslinked scaffolds (Scaffold14, 15, and 18). Since the elected SA:PVA ratio has been the same for all scaffolds, thereby one of the scaffolds has been nominated as the control group (without crosslinking). The SA/PVA electrospun scaffolds showed similar trends. The characteristic bands for SA were in the range of 3600 and 1500 cm−1. The characteristic bands of the scaffolds spectrum (Fig.5) are as follows. Appeared peaks at 3291 cm-1 and 2913 cm-1 belong to O─H stretching (hydroxyl group) and C─H stretching vibration respectively [54]. Appeared peak at 1088 cm-1 belongs CN group. The appeared sharp peak at 1717 cm-1 is attributed to the carboxylate group [55]. Comparing with the control group, a shoulder appeared before the peak at 1087 cm-1 and expanded the appeared peak. Besides, a new peak appeared at 943 cm-1 belongs to the CH2 rocking [56] which probably is attributed to the process of cross-linking by glutaraldehyde [57]. The spectra of the scaffolds were similar to that of pure PVA [58] which the reason is the high PVA content in all scaffolds (SA:PVA; 1:6.5). the appeared peak at 843 cm-1 is attributed to the C─C stretching [59].
3.4 Degradation
The degree of destruction of each scaffold was also measured by observing a change in the mass of the samples after immersion in PBS over time. Fig.6 depicts the degradation behavior of the scaffolds during incubation. Scaffolds 14, 15 and 18 showed 28%, 33%, and 39% of degradation after 21 days of incubation in PBS under similar patterns. The low and high rate of degradation belonged to Scaffolds 14 and 18 respectively. Various reasons can influence degradation behavior. Although the scaffolds experienced the same conditions in the cross-linking process there is a likelihood of variance in the level of cross-linking. However, the results from the FTIR did not show significant differences in cross-linking and chemical structures. Ki-Taek Lim et al. [60] reported that cross-linker and the time of cross-linking could affect the degradation process. By the way, the changes in the electrospinning parameters lead to a difference in density and diameter of nanofibers. The more nanofibers, the more chemical band between the polymeric chains [61]. Based on the results from porosity and SEM, it was hypothesized that the higher porosity can be imagined as the vital parameter in the rate of degradation behavior. The higher porosity resulted in low density and also cross-linking [62].
3.5 Swelling
The swelling behavior of the scaffold demonstrates the ability of nutrients and wastes to exchange between the environment and cells embedded in the scaffold to produce artificial tissue. Swelling efficiency directly refers to the ability to hydrate and stabilize within the biological systems [63, 64]. All scaffolds were incubated in PBS to evaluate the rate of water absorption over time.
The behavior of scaffolds in water absorption and swelling showed similar trends (Fig. 7). Scaffolds 14, 15, and 18 revealed 250%, 260%, and 160% swelling respectively after 24 h of incubation. According to the data, Scaffold 14 and 15 showed the highest but closed water absorption in contrast with another scaffold 18. The reason may be attributed to the high porosity of the scaffold 18 which means the low density of nanofibers [65]. It has been reported that the swelling potential of the scaffolds can be affected by the degree of crosslinking, amorphous regions, and level of hydroxyl groups [66-68]. According to the FTIR results, due to no significant difference in the results, it seems that the degree of cross-linking did not affect swelling notably. Comparing all scaffolds, they are made of the same SA/PVA combination but were different in operating parameters. Nanofiber diameter is one of the vital parameters of electrospun scaffolds and is affected by surface tension, solution viscosity, working distance, flow rate, crystallization characteristics and applied voltage [69]. Nanofiber diameter also can affect the porosity of the scaffold, thereby it could be concluded that porosity has been affected by operating conditions [12]. This effect may appear in the density of nanofibers per 1 cm2 and the diameter of nanofibers (166-227 nm). Furthermore, it is hypothesized that thick nanofibers will adsorb a higher amount of water than thin nanofibers. So a dense scaffold is predicted to be degraded late.
3.6 Tensile Strength
The mechanical behavior is a critical factor in studying the mechanical behavior of a scaffold. The tensile strength (MPa) of the scaffolds was measured by determining the strain-stress curve and measurement of the elastic modulus (EM) of each scaffold Fig.6. Scaffolds 14 and 15 had closed trends (Fig.8-A) indicating no significant difference (P > 0.05) in the viewpoint of EM compared with the scaffold 18 (Fig8-B). However, scaffold 15 revealed higher EM compared with Scaffold 14 and 18. Scaffold 18 showed the lowest EM (P<0.05) (Fig.8-B). It has been approved that the porosity has an adverse effect on mechanical behavior [70]. It was also reported that cross-linking can be the main factor affecting the mechanical behavior of scaffolds [71]. Hence, according to Table 4, scaffold 18 owned higher porosity, which low EM can be predicted about it. In the viewpoint of nanofiber diameters, interestingly, reduction in nanofiber diameters caused an enhancement in mechanical response including Young's modulus (EM) and tensile strength which the surface confinement of chains on the distribution of stresses in the fibers was considered as the main reason [69]. Smooth nanofibers with uniform distribution of diameter result in the densely packed scaffold with a high-molecular orientation that leads to higher resistance to the axial tensile forces. In this regard, it was reasonable that scaffold 15 reveals higher EM.
3.7 MTT
This study aimed to fabricate a SA/PVA electrospun scaffold in skin TE; thereby, it was necessary to assess the cytotoxicity and biocompatibility of the scaffolds. The MTT assay was determined for suitability of scaffolds for Fibroblast L929 cell line viability, as shown in Fig. 9. According to the cytotoxic assay, there was no significant difference in viability (P > 0.05) between the scaffolds compared with the control group ( > 75%), which means that all three scaffolds are suitable for cell culture and skin TE purposes. Based on the results from swelling and porosity assessments, the diversity in porosity and nanofiber diameters did not make a significant difference in the cell viability of the scaffolds. On the other hand, based on cell growth as well as cell concentration in each scaffold, it can be claimed that all scaffolds showed good cell adhesion compared to other scaffolds. Previous studies reported the biocompatibility of SA, PVA and SA/PVA. For instance, wei and You-Lo fabricated SA/PVA hybrid fibers (40% PVA and 60 SA) under physical crosslinking. They reported that the nanofibers were biocompatible and showed no cytotoxicity [53]. Pure SA also showed higher biocompatibility and in spite of it higher potential in TE, still, no cytotoxicity has been reported for this Polysaccharide [72]. Regarding PVA, biocompatibility results from the previous studies demonstrated that pure PVA was slightly toxin and irritant to the surrounding tissues [73]. However, it was reported that PVA biocompatibility can be improved when integrated with other biocompatible polymers including collagen, SA, Gellatin, and so on [74].