Poly (vinyl alcohol) (PVA, Mw: 72 kDa), chitosan (CS, MW: 190–300 kDa, 75%–85% deacetylated), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and thymol (CAS No: 89-83-8) were obtained from Sigma-Aldrich (Steinheim, Germany). Acetic acid and ethanol were bought from Merck Company (Darmstadt, Germany).

The blank PVA/CS nanofibrous scaffold and the scaffold containing thymol were fabricated with the electrospinning technique. To prepare the blank scaffold, a mixture of PVA (10% w/v) and CS (2% w/v) was blended at the ratio of 80:20 v/v. Then, the polymeric mixture was subjected to electrospinning at room temperature (25°C), set at +20 kV applied voltage, flow rate 1 mL/h, and a 15 cm tip-to-collector distance. Likewise, thymol (5–15 wt%) was added to the PVA/CS blended solution and was converted to the electrospun fibers at the same condition. After that, they were removed from the aluminium sheet and stored for further characterizations and examinations.

Scanning electron microscopy (S-4700, Hitachi Ltd., Tokyo, Japan) at an accelerating voltage of 30.0 kV after a gold plasma sputtering characterized the scaffolds’ morphology and diameter distribution. Besides, studying the interactions between the scaffold ingredients and endorsing the incorporation of thymol was conducted using Fourier transform infrared (FT-IR) spectrophotometer (IR Affinity 1S, Shimadzu, Japan) from 400 to 4,000 cm⁻¹ at room temperature. The water absorption capacity and the surface wettability of the scaffolds were determined by immersion in water (pH 7.4) at 37°C and water contact angle measurement through the sessile drop technique, respectively (Doostan et al., 2023). Moreover, the antioxidant activity of the prepared scaffolds was performed via DPPH radical scavenging assay as previously described (Mastelić et al., 2008).

Furthermore, thermal analysis was conducted using Differential Scanning Calorimetry (DSC) to elucidate the thymol-polymer interactions and structural transitions (Zeugolis and Raghunath, 2010). The samples, including pure thymol, blank scaffold, and loaded scaffold, were analyzed with a thermal analyzer (NETZSCH STA 449 F3 Jupiter, Germany) under a nitrogen atmosphere from 25°C to 100°C at a rate of 10°C/min.

In the study, 42 adult male Wistar rats weighing (220–260 g) were bought from Aftab Lorestan and held in the central animal house, at Kermanshah University of Medical Sciences. Animals were fed ad libitum and kept under standard conditions (light/dark cycle for 12 h in relative humidity of 60% ± 5% and temperature of 24°C ± 2°C). Procedures were performed following the policies for the treatment and care of laboratory animals published by The Iranian National Institute of Health and approved by the Ethics Committee at the Kermanshah University of Medical Sciences (Ethical code: IR.NIMAD.REC.1400.108). All actions were made to relieve mouse distress.

Rats were randomly split into seven groups each six rats as follows: 1) Group 1 (Sham group): received laminectomy without injury and were treated with normal saline; 2) Group 2: received compression injury; 3) Group 3: received compression injury and were treated with thymol (5 mg/kg, intraperitoneally); 4) Group 4: received compression injury and were treated with just the blank scaffold (in-situ); 5) Group 5: received compression injury and were treated with the nanofibrous scaffold containing 5 wt% thymol (in-situ); 6) Group 6: received compression injury and were treated with the nanofibrous scaffold containing 10 wt% thymol (in-situ); 7) Group 7: received compression injury and were treated with the nanofibrous scaffold containing 15 wt% thymol (in-situ).

Animals were transferred to the laboratory before testing to be adapted to the environment. Anesthesia was induced in rats, by the mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg) injected intraperitoneal (IP). The surgical area was shaved and disinfected with ethanol 70%. Laminectomy was accomplished at the T8-T9 level with a micro rongeur (Fine Science Tools, United States). Henceforward, the extradural clip compression SCI caused damage by closing an aneurysm clip (Aesculap, Tuttlingen, Germany) with a 90 g calibrated closing force, near the spinal cord for 1 min (Poon et al., 2007; Cheriyan et al., 2014; Bagheri Bavandpouri et al., 2024). Then muscles and skin were sutured, and rats were permitted to recover on a 30°C heating pad, and then received saline (2 mL, twice a day, subcutaneously) and cefazoline (40 mg/kg twice on the day of surgery, i.p.) to rehydrate and control urinary tract infections. SCI rendered all animals thoroughly paraplegic. Urinary excretion was manually dragged out two times per day until achieving bladder-emptying reflex comeback.

The rats were weighed at the commencement of experiments and repeated on days 7, 14, 21, and 28. The weight changes in each group were calculated through the directions below:

Weight difference = (Animal’s weight on days 7, 14, 21, and 28 – weight on day 0 (before the surgery)).

Basso, Beattie, and Bresnahan (BBB) is a locomotor behavior test that is a proper and predictive estimation of locomotor recovery and can also differentiate behavioral developments due to separate injuries (Basso et al., 1995). The approach was based on evaluated behavior before the injury, on the first postoperative day, and then for up to 4 weeks on days 1, 7, 14, 21, and 28. Scoring categories were determined and classified based on the experimental ordering of the Basso, Beattie, and Bresnahan Locomotor Rating Scale.

The von Frey test is a gold standard for specifying mechanical thresholds, which were invented by Maximilian von Frey and evaluating mechanical allodynia in mice and rats (Deuis et al., 2017). In this test, a monofilament was used vertically to the plantar surface of the rat’s rear paw until its apex, producing a stable pre-determined force (typically 15–180 g for rats) for 2–5 s. When any quick paw withdrawal, licking, or shaking was observed while applying the stimulus, it was considered a positive response. The test was repeated on days 7, 14, 21, and 28.

Cold chemical and thermal sensitiveness were estimated by utilizing the acetone drop method (Yoon et al., 1994; Ruan et al., 2018). The technique involved applying (100 μL) of acetone using a micropipette to the rat’s hind paw through the wired mesh cage. The rats were placed in the cage and allowed to acclimate for approximately 20 min before acetone application to facilitate exploration of their surroundings. The acetone drop was applied mildly to the rat’s rear paws. Rats’ reactions such as paw licking, shaking, or rubbing the hind paw and brisk foot withdrawal typically 2–5 s after the acetone administration were documented as positive responses. For each measurement, the paw was sampled three times with an interval of 5 min calculated, which scored 0, 1, 2, 3 and 4, respectively. The test was repeated on days 7, 14, 21, and 28.

The hot-plate test scored behavioural for measurements to estimate acute pain sensitivity (Hunskaar et al., 1986). Rats were put, each one separately, on a surface maintained at 50°C–56°C. The rat’s movement was restricted by a plexiglass cylinder. The latency to hind paw licking, shaking, or jumping, was recorded by time, and at this point, the animal was instantly released from the hot plate to lessen the suffering. The test was repeated on days 7, 14, 21, and 28.

All rats were sacrificed (100 mg/kg ketamine + xylazine 20 mg/kg) at the end of day 28 when the behavioral tests were finished. The spinal cord tissue was attentively extracted and then kept in 200 mL of 4% paraformaldehyde and 0.1 M phosphate-buffered saline (PBS). The tissues were then processed (Didban sabz DS2080/H; Iran). The sections of the spinal cord in each rat were stained with H&E. Histopathological alterations were analyzed using a light microscope, and the data was collected at a magnification of 40X.

Statistical analyses were performed using GraphPad Prism software version 6. Results are presented as mean ± standard error of the mean (SEM). One-way or two-way analysis of variance (ANOVA) was conducted, followed by Tukey’s or Bonferroni post hoc tests, to determine significant differences between groups. A p-value of less than 0.05 was considered statistically significant (Fakhri et al., 2018; Bagheri Bavandpouri et al., 2024).

The experimental design is schematized in Figure 1.

The SEM micrographs of blank electrospun PVA/CS scaffold and PVA/CS scaffolds containing 5-15 wt% thymol are depicted in Figure 2. The blank scaffold (without thymol) indicated the diameter distribution 313 ± 45 nm and the uniform morphological structure with the smooth, cylindrical, ribbon-shaped, and bead-free NFs with high porous structure (Figure 2A). Adding 5 wt% thymol in the PVA/CS mixture led to increasing the diameter of NFs to 390 ± 45 nm without any change in the morphology (Figure 2B). Likewise, the uniform and beadless NFs with a diameter distribution of 396 ± 49 nm were formed while the thymol concentration was augmented to 10 wt% (Figure 2C). Whereas, adding 15 wt% thymol resulted in the elevation of the average diameter to 412 ± 53 nm with appropriate morphology (Figure 2D). Further incorporation of thymol (>15 wt%) led to the formation of non-uniform NFs in diameter and morphology that is not desirable and was not used.

FT-IR spectroscopy was performed to verify the chemical structure of samples and identify the presence of thymol and its interaction with the polymeric matrix. The FT-IR spectra of pure thymol, the unloaded (blank), and the thymol-loaded scaffolds are presented in Figure 3. The spectrum of thymol indicated the distinguishing peak at 806 cm⁻¹ corresponding to out-of-plane C–H wagging vibrations, and peaks at 1,089 cm⁻¹, 1,244 cm⁻¹, 1,450 cm⁻¹, and 1,602 cm⁻¹ are attributed to C–O, C–C and C=C stretching vibrations of the phenolic ring of thymol (Wu et al., 2021). In addition, the characteristic peaks occurring at 2,960 cm⁻¹ and 3,202 cm⁻¹ are assigned to methyl groups (–CH₃) and hydroxyl groups (–OH) of thymol, respectively (Sanchez-Rexach et al., 2016; Michalska-Sionkowska et al., 2017). Besides, the spectrum of blank PVA/CS nanofibrous scaffold exhibited characteristic peaks at 1,080 cm⁻¹ and 1,251 cm⁻¹ that are associated with C–O–C stretching of ether groups as well as a sharp peak at 1728 cm⁻¹ corresponding to the C=O stretching bond of the residual acetyl groups in the CS (Rukmani and Sundrarajan, 2012). Also, the absorption peak at 2,935 cm⁻¹ is related to the–CH₂ group, and the broad peak at around 3,327 cm⁻¹ is related to the–OH/–NH₂ vibrations of PVA and the primary amines of CS (Rukmani and Sundrarajan, 2012). Furthermore, the spectrum of thymol-loaded PVA/CS NFs elucidated the prominent peaks similar to both thymol and PVA/CS NFs, confirming thymol’s presence in the NF structure without any adverse interaction as well as increasing intensity of peaks explicates the efficient loading into the nanofibrous scaffold.

The water absorption capability of the prepared scaffolds is depicted in Figure 4A. In the 30-min immersion, the water holding capacity of the blank PVA/CS scaffold was about 258% and reached 603% after 24 h immersion. The thymol-loaded scaffolds (5, 10, and 15 wt%) were able to absorb nearly 248%, 249%, and 239% reached 591%, 282%, and 575% after 24 h immersion, respectively. After 24 h, no change in the amounts of absorption was observed. These findings signify the high and rapid water absorption ability of the scaffolds, which is probably related to their high porosity. Further, the incorporation of thymol did not significantly alter the PVA/CS scaffold’s absorption capability, and there was no significant difference between the scaffolds.

Furthermore, the surface wettability of the scaffolds was evaluated through the water contact angle shown in Figure 4B. The measured contact angle of the blank PVA/CS scaffold was about 38.7°. While these values were 70.2°, 73.6°, and 76.9° for the loaded scaffolds containing 5, 10, and 15 wt% thymol, respectively. These results indicate the prepared scaffolds have a highly hydrophilic and wettable surface, affecting biological response, protein adsorption, and cellular attachment (Doostan et al., 2023).

The DSC thermograms of pure thymol and the prepared scaffolds are displayed in Figure 5. In the DSC curve of thymol, a sharp endothermic peak appeared around 57°C, corresponding closely to its melting point (Zamani et al., 2015; Giotopoulou et al., 2024). The blank PVA/CS scaffold did not exhibit sharp peaks, indicating the absence of crystalline microstructure. The majority of polymeric chains in the non-crystalline state in the electrospun NFs is likely due to the rapid solidification of stretched polymeric chains during the electrospinning process (Jia et al., 2007; Gholipour, K et al., 2009; Olvera Bernal et al., 2023). Likewise, the DSC thermograph of the thymol-loaded scaffold indicates that the characteristic melting endothermic peak of thymol disappeared upon incorporation into the electrospun NFs. This result implies that thymol converts from crystalline to amorphous form, presumably due to rapid solvent evaporation and restricted molecular mobility caused by intermolecular interactions, e.g., hydrogen bonds, during electrospinning (Chen et al., 2020; Jiffrin et al., 2022). Such conversion, leading to the molecular dispersion of thymol during incorporation into nanofibrous scaffolds, can provide homogeneous distribution and higher drug loading in the nanofiber matrix, ensuring consistent release profiles, which is advantageous for poorly soluble drugs, enhancing solubility, bioavailability, and dissolution rate (Qi and Craig, 2016; Jiffrin et al., 2022).

The antioxidant activity of the scaffolds was determined by measuring the DPPH free radical scavenging strength; the results of which are shown in Figure 6A. The blank scaffold showed low antioxidant activity (17%) at high concentrations, possibly owing to the absorption of DDPH molecules by the large surface area of the NFs. The antioxidant activity of the scaffolds was augmented significantly by adding thymol. The activities of the scaffolds rose significantly by adding thymol and further increased in higher quantities. At concentrations of 25–400 μg/mL, the scavenging activities of the scaffolds containing 5, 10, and 15 wt% thymol were found to be from 6%–61%, 15%–75%, and 22%–86%, respectively. The higher loading of thymol resulted in higher antioxidant activity that is due to the presence of thymol in the scaffolds, enabling to donate of hydrogen by hydroxyl groups in the structure and breaking the chain reactions of free radicals (Aziz and Almasi, 2018). Scaffolds exhibited a concentration-dependent activity for radical scavenging, and a rise in thymol content increased antioxidant activity. Blank nanofibrous scaffold of PVA/CS exhibited no radical scavenging activity, but 5, 10, and 15 wt% thymol-loaded scaffolds exhibited significantly increased antioxidant activity at all concentrations (p < 0.05). To further evaluate the antioxidant efficacy, a half-maximal inhibitory concentration (IC₅₀) value was determined with a sigmoidal fitting model Figure 6B. The IC₅₀ values, representing a 50% free radical-scavenging activity, were calculated as: 5 wt% thymol-loaded scaffold: 264.52 µg, 10 wt% thymol-loaded scaffold: 194.93 µg, 15 wt% thymol-loaded scaffold: 102.14 µg. These results confirm that the 15 wt% thymol-loaded scaffold exhibited the highest antioxidant activity, with its lowest IC₅₀ values. Dose-dependent enhancement in free radical-scavenging activity confirms that thymol is a key player in contributing towards the antioxidant activity of nanofibrous scaffolds, and it can contribute towards minimizing oxidative stress in SCI.

The weight changes of the animals compared to the day before surgery were evaluated and are shown in Figure 7. The obtained results indicate that the sham group showed a normal weight gain pattern over the 4-week period. However, in the injured rats compared to the Sham group, a significant impairment in weight gain was observed during the weeks following the injury (p < 0.001). Treatment with thymol-loaded scaffolds at 10% and 5% resulted in a significant increase in the normal weight of the animals during the 21 days after the injury (p < 0.001), while thymol-loaded scaffolds at 15% led to a notable increase as well (p < 0.01) on day 21. This observed improvement eventually compensated for the weight loss caused by the injury, and no significant differences were observed between the treatment groups.

Motor performance of rats after SCI was measured by the BBB scale as shown in Figure 8. Examining the movement performance of the sham group indicated no movement disorders after laminectomy, and the rats consistently scored 21 on all examined days. In contrast, the SCI group exhibited complete paralysis on the first day after the injury, with scores of 0 and 1, and throughout the treatment period, they showed a significant decrease in motor performance compared to the sham group animals (p < 0.001). However, the groups treated with 10% and 15% thymol-loaded scaffolds showed an increase in the recovery of movements and joints by day 14 after the injury (p < 0.05), and there was a significant improvement in motor performance for the group treated with 5% and 10% thymol-loaded scaffolds on days 21 and 28 (p < 0.001).

Evaluation of mechanical pain was performed by applying the mechanical force of von Frey fibers on the dorsal surface of the rat’s hind legs, between the 2^nd and 3^rd toes (Figure 9). The obtained results show that in the sham group, the mechanical pain tolerance threshold remained consistent during the treatment period after laminectomy. On the other hand, in the SCI group, a significant decrease in the mechanical pain tolerance threshold was observed on day 7, and no significant changes were observed in the following days compared to the sham group (p < 0.001). The group treated with a 10% thymol-loaded scaffold demonstrated an improvement, showing a more pronounced reduction in neuropathic pain caused by foot injury on day 14 (p < 0.01), and continued to exhibit reduced pain levels on days 14, 21, and 28 (p < 0.001). Additionally, the group treated with 5% thymol-loaded scaffolds showed a reduction in neuropathic pain on day 7 (p < 0.05) and sustained the reduction on day 28 (p < 0.001).

The acetone drop test was used to assess the animals’ tolerance threshold to the cold pain stimulus and the data are depicted in Figure 10A. In the sham group, the foot withdrawal reflex in response to the cold stimulus (acetone) remained consistent, and no significant changes were observed in the days following laminectomy. This is despite the fact that the injured animals became highly sensitive to cold and their pain tolerance threshold decreased more clearly, also there is a significant difference in the comparison of the reflexes of this group with the Sham group (p < 0.001). Treatment with a 10% thymol-loaded scaffold increased the tolerance of the response threshold to cold stimulus from day 14 until day 28 after injury (p < 0.001) and also 5% thymol-loaded scaffold and blank scaffold increased the tolerance of the response threshold to the cold stimulus at day 21 (p < 0.001). In contrast, the 15% thymol-loaded scaffold showed a moderate increase at day 21 (p < 0.01).

Thermal pain was assessed using the hot plate test, as shown in Figure 10B. The obtained results indicated that the time delay in foot licking, as a measure of pain tolerance threshold due to heat in the sham group, did not change significantly from the first to the 28th days after laminectomy. Compared to the sham group, the SCI group showed a much lower tolerance to thermal pain on days 7 and 14 (p < 0.001) and in the last 2 weeks, the pain tolerance threshold increased slightly in the direction of recovery (p < 0.001). Treatment with a 5 mg/kg dose of thymol and 10% thymol-loaded scaffold was associated with a moderate increase in the tolerance threshold of response to thermal stimuli with a relatively mild slope in the final week after injury (p < 0.05).

Hematoxylin-eosin staining was used to investigate the extent of tissue damage after SCI, as depicted in Figure 11A. Counting the number of motor neurons (Figure 11B) in the area of the ventral horn of the gray matter of the spinal cord on day 28 after the injury revealed a significant decrease in the number of neurons at the injury site (p < 0.001). Treatment with 10% and 5% thymol-loaded scaffolds demonstrated improved protection of neurons and led to an increase in the number of motor neurons in the ventral region of the spinal cord (p < 0.001). Similarly, 15% thymol-loaded scaffold treatment also showed improvement in the number of motor neurons (p < 0.01).