SHEDs were obtained from the Stem Cells Technology Research Center of the University. They were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin; Gibco) at 37°C and in 5% CO₂ atmosphere. At approximately 80% confluence, SHEDs at passages 2–4 were washed with phosphate buffer saline (Gibco) and transferred to a serum-free DMEM culture medium. After 48 hours, the CM was harvested and centrifuged at 1,500 rpm for 5 minutes. The supernatant was recentrifuged at 3,000 rpm for 3 minutes, followed by collection of the second supernatant. The CM was concentrated 10 times using ultrafiltration with a cut-off of 10 kDa (Millipore, Bedford, MA, USA) and stored at −80°C [8-10].

Collagen was extracted from the rat tail based on the instructions provided in a previous study [15]. It was dissolved in 0.6% acetic acid at 4°C to obtain a concentration of 4 mg/mL. Next, 5 mL of the prepared solution, 5 mL of DMEM or SHED-CM, and 1 M NaOH were mixed in that order to obtain collagen solutions at pH 7.5 and a temperature of 4°C. Final solutions containing DMEM or SHED-CM were used for injections.

Adult male Sprague-Dawley rats weighing 250–280 g were used in the present research. The animals were housed in standard conditions. The research was performed with the approval and guidelines of the Animal Care and Ethics Committee of the University (approval no., 95-11655).

The rats were randomly divided into the following five groups (n=10/group): (1) control: laminectomy was performed; (2) SCI: laminectomy and SCI were performed; (3) SCI+SHED-CM: laminectomy and SCI were performed, and SHED-CM was injected into the injury site; (4) SCI+Col: laminectomy and SCI were performed, and collagen hydrogel was injected into the injury site; and (5) SCI+SHED-CM+Col: laminectomy and SCI were performed, and SHED-CM loaded in collagen hydrogel was injected into the injury site.

Surgical procedures were performed under deep anesthesia with ketamine (80 mg/kg) and xylazine (10 mg/kg). In summary, laminectomy was performed at the thoracic level, and a compression injury was induced at T7 level by the application of a 20 g aneurysm clip (Harvard Apparatus) for 1 minute. Immediately after induction of SCI, the determinate biomaterials for each group were injected into the injury epicenter (at a depth of 1.5–2 mm from the dura) using a 33-gauge Hamilton syringe for a total dose of 3 μL. The needle was maintained in the cord for 5 minutes to prevent reflux; then, it was ejected, and the muscles and skin were sutured. After surgery, the animals were kept in a warm environment until regaining consciousness. A combination of tylosin and meloxicam was immediately injected after the surgery, followed by three additional injections for 1 day post-injury for pain relief and prevention of infection. The rats’ bladders were manually expressed 3 times daily until the return of spontaneous bladder function [16].

Behavioral tests were classified as locomotor, motor, sensory, and sensory-motor to examine different aspects of behavioral performance [3]. The time of the day when the tests were performed and order of testing remained the same during the experimental period. A 30-minute interval was considered as resting time after performing different tests. Testing conditions were constant for all the tests and animals. Testing devices were cleaned after each trial.

Locomotor performance was assessed using the Basso, Beattie, and Bresnahan (BBB) open-field test. Animals were acclimatized to the testing equipment and environment. All animals were examined before the surgery, on day 2 post-surgery, and then weekly up to 6 weeks. BBB testing was used to measure specific components of locomotor ability, including hind limb movement, trunk stability, toe clearance, and body, tail, and paw positions. The BBB rating scale ranged from 0 to a maximum of 21. A score of 0 indicated no observable locomotion, and 21 indicated normal locomotor activity. All the animals were evaluated for 5 minutes by two experienced observers. The mean scores recorded from both hind paws were used for analysis [17].

The inclined plane test was performed to assess motor function by observing muscle strength of the hind paw. All the animals were evaluated before the surgery, on day 2 post-surgery, and then weekly up to 6 weeks. The animals were placed on an inclined plane. The inclined plane was incrementally raised from the horizontal plane (0°), and the largest angle at which the animal could maintain its position for 5 seconds was recorded. Three measurements were performed for each animal, and the mean score was used for analysis [18].

Animals were administered the cold allodynia test. Foot withdrawal reaction to acetone contact was considered as cold sensitivity. All the animals were assessed before the surgery, on day 2 post-surgery, and then once weekly up to 6 weeks. Before starting the trial, animals could habituate inside the test apparatus (transparent cage on metal mesh) for approximately 10 minutes. A fresh acetone drop (50–100 μ) was lightly dabbed to the mid plantar surface of both the hind paws using a syringe. It was repeated for five administrations with a minimum of 5 minutes rest between trials. Either brisk foot withdrawal or flinching was regarded as a positive reaction. The time spent for responding was measured for each animal [19].

Animals were assessed for the sensory-motor function using the beam walk test. Assessments were performed before the surgery, at 2 weeks post-surgery, and then once weekly for up to 6 weeks. All the animals were tested on seven beams with different widths. A scoring scale, ranging from 1 to a maximum of 7, was used according to the beam width. Rats were placed on the beams sequentially from the widest to the narrowest. Two trials were performed for each rat. The beam with the narrowest width that an animal was able to walk without foot slips was recorded [20].

The data are presented as the mean±standard deviation. Analysis of variance followed by Tukey’s post-hoc test was used to compare the mean values using the IBM SPSS ver. 19.0 statistical software (IBM Corp., Armonk, NY, USA). All p≤0.05 were considered statistically significant.

All the groups had BBB scores of 21 before injury, indicating normal locomotion (Fig. 1, Table 1). The control group showed a BBB score of 21 throughout the study. There were significant differences between BBB scores of the control and other groups at all time points post-surgery (p<0.001). All injured animals presented flaccid hind limb paralysis with a BBB score of approximately 0 on day 2 post-surgery. Over the following 6 weeks, continuous recovery of hind limb locomotion was observed in all injured animals. However, BBB scores of the SCI+SHED-CM+ Col group were significantly higher than those of the other groups beginning at 1 week post-surgery, and the difference remained significant throughout the study (p<0.05, p<0.01) (Fig. 1, Table 1). Notably, no significant differences were found among SCI+SHED-CM, SCI+Col, and SCI groups. BBB score of 9 is critical to indicate a weight-supporting step. Fig. 1 shows that this stage was reached 4 days earlier in the SCI+SHED-CM+Col group compared with other injured groups (Fig. 1, Table 1).

The mean angle recorded in the inclined plane test before injury was approximately 70° for all the animals (Fig. 2, Table 2). The control group maintained this result throughout the study. Significant differences were observed between the control and other groups at all time points post-surgery (p<0.001). After surgery, a continuous increase in the mean angle recorded in the inclined plane test was observed in all injured animals. The SCI+SHED-CM+ Col group showed a significantly better performance than that by other injured groups at 2 weeks post-surgery and at all subsequent time points (p<0.05, p<0.01) (Fig. 2, Table 2). No significant differences were found among the SCI+SHED-CM, SCI+Col, and SCI groups.

One of the distinctive features of clinical neuropathic pain is increased sensitivity to typically nonpainful cold stimuli. In our study, SCI induced adverse pain behavior of cold allodynia with positive reactions to a cold stimulus. Sensitivity to acetone contact increased at 2, 3, and 4 weeks post-injury in all the injured groups. Afterward, a decrease in sensitivity was observed at 5 and 6 weeks post-injury. The duration of response to acetone was significantly lower in the SCI+SHED-CM+Col group than in the other injured groups at 2, 3, and 4 weeks post-injury, indicating lower sensitivity (p<0.05, p<0.01) (Fig. 3, Table 3). There were no significant differences among the SCI+SHED-CM, SCI+Col, and SCI groups. The duration of response to acetone was constant in the control group throughout the study. In addition, at the end of the study, no significant differences were observed among all the groups, indicating recovery of all animals (Fig. 3, Table 3).

Before injury, all rats were able to cross the beam with the narrowest width without any errors in foot placement (Fig. 4, Table 4). The control group showed a maximum beam walk score of 7 throughout the study. All injured groups showed severe deficits following injury. The beam walk score was significantly higher in the SCI+SHED-CM+Col group than in the other injured groups beginning at 4 weeks post-injury and during the subsequent weeks (p<0.05, p<0.01) (Fig. 4, Table 4). No significant differences were observed among the SCI+SHED-CM, SCI+Col, and SCI groups.