Preparation and Characterization of Sodium Alginate Polymeric Scaffold by Electrospinning Method for Skin Tissue Engineering Application


 Sodium alginate (SA) approved its high potential in tissue engineering and regenerative medicine. One of the main weaknesses of this polysaccharide is its low spinnability which nanofiber based scaffolds are the interest of scientists in biomedical engineering. The main aim of this study was to improve the spinnability of SA in combination with polyvinyl alcohol (PVA). It was also tried to optimize the main parameters in electrospinning of the optimized SA;PVA ratio including voltage, flow rate, and working space. To aim this, Response surface methodology under central composite design was employed to design the experiments scientifically. The final nanofiber scaffolds were studied using scanning electron microscopy, Fourier transform infrared spectroscopy, degradability, swelling, tensile strength, porosity, nanofiber diameter, contact angle, and cytotoxicity. Based on the results, the best ratio for SA:PVA was 1:6.5. The solution with this concentration was spinnable in various values for the process parameters. The fabricated scaffolds under these conditions revealed good physical, chemical, mechanical, and biological features. L929 cell lines revealed high viability during 48 h of culture. The results revealed the uniform and homogeneous nanofibers with the regular size distribution (166 nm) were obtained at 30 kV, 0.55 µl/h, and 12.5 cm. To sum up, the optimized ratio under the reported conditions can be a good biologically compatible candidate for skin tissue engineering.


Introduction
Tissue engineering (TE) is growing as a novel biomedical engineering area to redevelop newfangled material for substituting problematic or injured tissues [1,2]. It comprises the construction of natural and/or synthetic structures, allows the combination of these materials with growth factors and/or signaling molecules to modulate cell proliferation and differentiation, and develop constructs mimicking the extracellular matrix (ECM) [3].
TE of skin substitutes signi es a potential foundation of improved treatment in ghting acute and chronic skin injuries [4]. Human skin is the widest organ of the body affected by injuries such as infection, burns, and disease [5]. There are no signi cant prototypes of engineered skin duplicate the composition, structure, organic constancy, or visual environment of healthy skin. Skin alternates should carry some essential physiognomies that comprise being simple to use [6].
Recent advances in skin TE have offered the potential to improve skin regeneration's clinical outcome [7,8]. However, some de ciencies need to be addressed to provide substitutes with the painless and rapid healing process and encourage vascular, neural, and lymphatic networks, hair follicles, sebaceous and sweat glands [9]. Therefore, skin TE's ultimate goal is to fabricate a complicated scar-free skin substitute that can be transplanted in large quantities in only one surgical intervention with a minimum chance of rejection by the host's body [10,11].
One of the main factors that in uence graft success is the scaffolding technique. Some of the main criteria for designing a scaffold are cell adhesion, in ltration, proliferation, and differentiation, and capable of creating new tissue [12]. Various techniques have been reported for skin TE, including 3D printing, electrospinning, freeze-drying, gas foaming, etc. Scaffolds fabricated by electrospinning have been classi ed as an optimal scaffolding option with bene cial biological and mechanical properties [13,14]. Electrospun nano bers have exceptional properties such as a similar structure to the natural extracellular matrix (ECM) [15], permeability [16], and scar formation regulation [17]. This technique has been used extensively in the eld of skin TE and various natural and synthetic biomaterials such as PCL [18], poly (lacto-co-glycolic acid) (PLGA) [19], polyvinyl alcohol (PVA) [20], sodium alginate(SA) [21], Bacterial cellulose [22], chitosan [23], and collagen [24] have been utilized to fabricate electrospun scaffolds (nicely reviewed by Quynh P. Pham [25]). Among them, SA and PVA were considered in tissue engineering.
There are several studies considering blends of SA and PVA for TE purposes. In research by Manikandan,G.,et al. [26], scientists approved that SA/PVA composition can be a suitable candidate for liver TE. Liver cells had excellent adhesion. In the case of bone TE, SA/PVA 3D printed scaffold revealed its high potential in cell viability due to having homogeneous porosity and improved hydrophilic properties. The scaffold had excellent mechanical properties, and its modulus of elasticity showed promising results [27].
Similarity Coelho et al.[28] Showed that among the many polymer-based scaffolds fabricated for TE engineering, SA/PVA scaffolds are known to provide mechanical stability (high tensile strength and elongation at break), exibility and slow degradation kinetics to the scaffolds. Also, Alhosseini et al. [29] Showed that in neural tissues, the scaffold microstructure, its three-dimensionality, and aligned bers is as essential as its biological properties. Even though many materials and techniques have been employed in TE, SA/PVA based electrospun nano bers have been shown to meet all the requirements.
They can be tuned to t speci c alignments, porosity, and architectures while maintaining their exibility, mechanical properties, and biological features. In research by Vig et al. [30], SA/PVA blend was used to fabricate electrospun scaffold for skin regeneration. The made scaffold revealed good mechanical properties, hydrophilicity, cell attachment, and cell growth. In another research in skin regeneration, SA/PVA scaffold showed that that the presence of SA inside the cross-linked polymeric network improved the active substance delivery properties of the hydrogel lms. When more signi cant SA levels were applied, the hydrogel had an irregular surface structure, as revealed by SEM images.
According to the previous studies, the SA/PVA scaffold can be concluded depending on SA: PVA ratio has been nominated for both hard and soft tissues. Enhancement in SA content makes the scaffold suitable for soft tissues while increasing PVA content makes it eligible for hard tissues. Thereby, it is hypothesized that this blend can also be nominated for skin TE. Hence, the main aim of this study was to increase SA electri cation capability by using PVA to fabricate a new electrospun SA/PVA scaffold capable of supporting the skin broblast cell for skin TE.

Chemicals
Sodium alginate (SA, Sigma-Aldrich Canada Ltd, with a molecular weight of 216.12 g/mol) and polyvinyl alcohol (PVA, 99%, Merck), and glutaraldehyde were purchased from a local supplier, TemadKala Co., Tehran, Iran. All the materials and the reagents were in analytical grade.

Procedure
In this research, it was tried to fabricate electrospun SA-based scaffold by optimization and characterization of the nal formula and the main parameters in electrospinning including Pressure (P), Temperature (T), and Velocity (V). To aim this, as the step#1, rst the optimized formulation of SA and PVA was determined. Then, as the step#2, the optimized formula was employed to evaluate the optimized conditions

Design Expert(DOE)
In this study, Response surface methodology (RSM) using central composite design (CCD) was employed to nd the optimum formulation to prepare 3D printed SA/PVA scaffold with proper strand diameter, appropriate tensile strength, and high cell compatibility. The main parameters including P, T, and V were evaluated upon the optimized formulation. Accordingly, the percentage of PVA and SA in bioink composition were considered as the process parameters in DOE. Three levels, including low (−1), medium (0), and high (+1), were de ned for PVA and SA concentration separately. According to our literature study, for PVA, the low and high levels were 1% and 12% w/w, respectively, and for SA were 1% and 4%.
According to Table 1, 13 runs were performed. Printability was measured as the response. The measured response was transferred in the software, which provided equation and relevant graphs to show the governed relation between material composition and the considered response. The main aim of DOE was to nd out the most optimal condition and composition for making scaffold.

Polymeric solution preparation
In order to prepare the polymer solutions accurately, since both SA and PVA are water soluble, deionized water was used as solvent. The desired volume was considered 20 ml for each sample. First, the required amount of each substance was weighed according to the DOE results and then transferred to a 50 cc test tube and increased to a volume of 20 ml using deionized water. The tube was placed on a stirrer and the resulting solution was mixed for 12 hours. The nal solution was sonicated at 170 watts in an ultrasonic bath for 10 minutes. Finally, the samples were stored at refrigerator.

Electrospinning
Each sample was sonicated for 10 minutes before starting the electrospinning process. Then, 5 cc of each sample was transferred into a 10 cc syringe. It was noted that the solution be free of any bubbles.
The drums were covered by aluminum foil. The electrospinning process was investigated by changing the three parameters of voltage (<30 kV), ow rate (<1 ml/h), and the nozzle distance (<30 cm) from the drum.

Cross-linking
Since both polymers polyvinyl alcohol and sodium alginate are water-soluble, after the ber production process and drying, this solubility is still high, and on rst contact with the aqueous medium, the bers dissolve in water (culture medium). The cross-linking process was carried out to improve this issue. In this regard, 25% glutaraldehyde solution was used. For this purpose, the desired pieces were cut from foil and placed in a petri dish. 2 ml of 25% glutaraldehyde solution was poured into a small container and transferred to the petri dish containing ber pieces. The petri dish was sealed with para lm and were placed in an incubator at 37.5 ° C for 24 hours. At the end of the course, all glutaraldehyde solution was evaporated.

Scanning Electron Microscopy
To measure the size distribution and surface structure of the 3D printed scaffolds, and cell attachment, scanning electron microscopy (SEM) (Philips XL30; Philips, Eindhoven, Netherlands) was carried out under a 25 kV accelerated voltage after sputtering a gold layer with a 5 nm diameter on the samples. The average strand diameter was calculated using the ImageJ software (National Institute of Health, USA).

FTIR
To ensure the link between the SA and PVA functional groups and also the chemical bonds, speci c values of each sample were prepared and analyzed by infrared spectrometer (FTIR, SHIMADZU, 8400S model Japan) with KRS-5PRISM at a 45 degree angle. The IR spectrum appeared in the wavelength range 500 to 4000 cm -1 .

Degradation
Scaffolds were freeze-dried and then weighed to determine their initial masses. The samples were incubated in 10 mM PBS solution in pH=7.4 at 37 •C and 5% carbon dioxide (according to the cell culture conditions) for 3,7, 14, and 21 days to obtain the degraded scaffolds. The PBS solution was taken out of the samples and then washed with deionized water two times, and then samples were freeze-dried and weighed again using a digital scale. The scaffold degradation was calculated using the equation 1: is the freeze-dried scaffold weight at a given time, and 0 is the freeze-dried scaffold weight at the time zero.

Swelling
The primary weight of the scaffolds was measured after crosslinking. The scaffolds were then incubated in 10 mM PBS solution in pH 7.4 at 37 •C and 5% carbon dioxide (according to the cell culture conditions). The samples' weights were measured again after 24 h for any mass change due to swelling. A Kimwipe was used to eliminate excess or free liquid from the scaffolds before weighing each sample. The swelling of the composite scaffolds was calculated using the equation 2:

Contact Angel
To determine and compare the hydrophilicity of different scaffolds, the water contact angle of the samples was measured. For this purpose, rst the sample was placed on a at surface and then a drop of water was dropped on it with a moving needle. The spherical image of the droplet was transmitted to the monitor by a digital camera and then the contact angle of the droplet with the web surface of the nano bers was measured.

Porosity
The porosity of the scaffolds measured according to the SEM images using ImageJ software. In order to process the images to obtain the total porosity, the total porosity was measured as the sum of the areas between the bers, expressed as a percentage.

MTT
To evaluate the cytotoxicity of the prepared scaffolds, rst the electrospun scaffolds were immersed in 70% ethanol for 24 hours. After drying the scaffolds at room temperature, the scaffolds (both sides) were sterilized for one hour by exposure to UV rays. The scaffolds were then carefully placed on a plate and washed with sterile PBS. Fibroblast L929 cell line obtained from the cell bank in School of Advanced Technologies in Medicine (Shahid Beheshti University of Medical Sciences, Tehran, Iran) with a density of 2×10 3 per milliliter and were placed on scaffolds by drip method at a rate of 20 microliters. Next, the scaffolds were incubated for 48 h at 37°C and 5% CO 2 . At the end of the period, 10 μl of the MTT labeling reagent at the concentration of 0.5 mg/ml was added to each well and incubated the them for 4 h under the same conditions (37°C and 5% CO 2 ). Then, 100 μl of the solubilization solution was added into each well. Left the plate for incubation at 37°C and 5% CO 2 overnight. The purple formazan crystals were checked and the absorbance was measured by ELISA reader.

Result And Discussion
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 nano bers with better quality. To aim this, As can be seen in Table 1, 13 runs have been considered according to the RSM study to nd out the nano ber producibility of each formulation of SA and PVA. Table 2, represents 20 different conditions to produce nano bers. The DOE software provided quadratic equations as the governing relations between the percentage of ingredients and the selected response (nano ber production) were examined via ANOVA. Table 3 summarizes the results.
The reliability of a model is usually justi ed via P-value, which should be lower than 0.05 to conclude that the model tting the experimental data is valid and signi cant [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 nano ber producibility, P-value was higher than 0.05 for A and B (as the rst-order effects ), AB (interaction effect) and B 2 (as the second-order effects). P-value was lower for A 2 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 signi cance of the governed equation. The P-value was lower than 0.05 only for A as the rst-order effect, AB as the interaction order, C 2 as the second-order effect. P-value was too high for AC and BC and C.  [32]. both models showed R 2 equal 0.83 and 0.79 and had a reasonable agreement with adj.R 2 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 nano ber production and the likelihood of nano ber 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 nano ber 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 nally leads to the nano bers production [36]. For instance, Gong and his colleagues produced SA-based nano bers 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 nano bers. It was reported that the nal 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 nano bers. The mentioned ratio was considered as the optimized SA:PVA ratio. To analyze the nano ber and also the effect of process parameters on the quality of the synthesized nano bers, this ratio was used as the main formulation for the rest of the study. Other ratios did not result in nano ber production under any adjustment of operating parameters including voltage (0-30 kV), the working distance (5-20 cm), and ow 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 nano ber production negatively. To produce nano bers at low V, it is necessary to decrease the working distance (lower X). However nano ber 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 nano ber 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 ow 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 nano ber 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 nano ber 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 nano ber 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 nano bers [42]. It has also been reported that changing the applied voltage changes the quality of the nano bers, thereby changes the diameter and morphology of the nano bers [43]. Reneker et al. [44] stated that enhancement in the applied voltage has no effect on the ber diameter of PEO. However, in 2005 Zhang and his colleagues reported obtaining larger-diameter nano bers 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 nano ber diameter. Furthermore, it was observed that at higher applied voltages, bead formation was obtained on the bers [42,46].
Another parameter that affects the control of morphology and diameter of nano bers is the distance of the nozzle from the collector. To prevent evaporation of the polymer solution before the ber reaches the collector, it is necessary to optimize the distance [47]. Therefore, in the electrospinning method, an optimized distance is required to bers reach the collector and prevent solvent evaporation. Based on the results of our study, this distance depends entirely on the applied voltage and the ow rate. Longer distances have been reported to produce thinner bers [48] but this claim is true when increasing the distance does not have a negative effect on ber 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 bers need to be cooled to achieve uniform bers and to prevent ber fusion, shorter collector distances can increase the likelihood of ber fusion at the junction.
Polymer ow rate indicates the ow rate of the polymer solution per unit time, which is known as another factor affecting the quality of bers. It has been reported that increasing the ow rate causes more polymeric material to come out of the nozzle, which leads to the production of coarser bers. Low ow rates are essential for the production of good quality bers with uniform diameters [52]. It has been predicted that nano ber diameter decreases due to increased charge density at low rates [42]. It was also reported that with increasing ow rate, there is a continuous increase in nano ber diameter [44]. It is notable that an excessive increase in ow rate not only enhances the integration of nano bers but also creates a bead in the ber 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 magni cations and ber diameter distribution. The porosity and the ber diameters are also reported in Table 4. As can be seen, scaffolds depicted differences in nano ber density, uniform distribution, nano ber diameter, and the quality of electrospinning with fewer or no beads. Fig.3 shows that scaffolds 3 and 12 did not have uniform nano bers in size distribution and quality. The voltage was equal for both scaffolds but they were different in distance and ow rate. The scaffolds 2, 5, and 7 although had uniform nano bers but showed a low density of nano bers The reason can be attributed to the disproportion of ow rate to distance.
Low ow 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 nano bers and smooth bers. According to Table 4, the lowest and highest porosity belonged the scaffold 14 (521 nm 2 ) and 3 (1404 nm 2 ) respectively. Scaffolds 2 and 7 also had high porosity equal to 1004 and 1205 nm 2 respectively. Considering the size distribution of nano ber, Table 4 also shows that the thin nano ber belonged to the scaffold 12 and 15 (140-170 nm) while the scaffold 3 and 7 owned the thick nano bers (300±5 nm respectively). Scaffold s 2, 5, 14, and 18 showed nano bers 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 ow rate at 0.5-1 µl/h resulted in appropriate nano bers. The results were in agreement with previous studies. Hu and his colleagues produced SA/PEO nano bers 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 nano bers 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 5 meaning that the scaffolds are extremely hydrophlilic. According to the results from the morphology analysis and quality evaluation of the synthesized nano bers, Scaffold 14, 15 and 18 were nominated for more analysis. These scaffolds revealed appropriate density, smooth nano bers, uniform size distribution and suitable porosity. In the rest of the study, the scaffolds were rst crosslinked under 25% glutaraldehyde vapor and then were evaluated.

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 CH 2 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].

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

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 arti cial tissue. Swelling e ciency 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 . Nano ber 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 nano bers per 1 cm 2 and the diameter of nano bers (166-227 nm).
Furthermore, it is hypothesized that thick nano bers will adsorb a higher amount of water than thin nano bers. So a dense scaffold is predicted to be degraded late.

Tensile Strength
The mechanical behavior is a critical factor in studying the mechanical behavior of a scaffold. The 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 nano ber diameters, interestingly, reduction in nano ber diameters caused an enhancement in mechanical response including Young's modulus (EM) and tensile strength which the surface con nement of chains on the distribution of stresses in the bers was considered as the main reason [69]. Smooth nano bers 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.

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 signi cant 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 nano ber diameters did not make a signi cant 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 bers (40% PVA and 60 SA) under physical crosslinking. They reported that the nano bers 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].

Conclusion
The spinnability of Sodium alginate, a biodegradable and biocompatible polymer, was rst assessed in combination with the different percentages of PVA. Then the optimized SA:PVA ratio was nominated to optimize the processing parameters including voltage, working distance, and ow rate. SA inherently is not spinnable, thereby combination with other spinnable polymers improves its potential in nano ber production. Different percentages of PVA were studied and only the 6.5 PVA depicted good spinnability. The spinnability of the optimized ratio could be controlled with the variation of the applied voltage, ow rate, and working distance. some conditions did not result in nano bers. The results revealed the Uniform and homogeneous nano bers with the regular size distribution with narrow diameter (< 170 nm) were        cell viability analysis of the elected eletrospun scaffolds.