Sprague-Dawley (SD) female rats (8-weeks old, 180-250 g body weight) used in this experiment were obtained from SPF (Beijing) Biotechnology Co., Ltd. [permission number: SCX (JING) 2019-0010]. The rats were group-housed 2-3 per cage and maintained with a 12-h light/dark cycle (light at 7 a.m.) under a temperature (22-23˚C) and humidity (55-65%)-controlled environment. They were given free access to food and water and acclimated for at least 7 days before the surgery for the adaption to the environment. All procedures involving animals complied with the Guiding Principles for the Care and Use of Vertebrate Animals in Research and Training. The experiments were approved (approval no. MDL20210810-01) by the Animal Ethical and Welfare Committee of The Tianjin Medical University General Hospital (Tianjin, China).

The isolation, culture and identification of ASCs have been previously described (22). Briefly, an 8-week-old SD rat was anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg). To change SCs into ASCs, following anesthesia, the bilateral sciatic nerves were ligated for one week. Then the rat was sacrificed with intraperitoneal injection of excess pentobarbital (300 mg/kg) and the bilateral sciatic nerves distal to the ligation spot were isolated. After removing the endoneurium, the remaining nerve tissues were cut into 0.5-1 mm³ fragments. The nerve tissues were digested with 0.05% collagenase (cat. no. C8176; MilliporeSigma) for 45 min at 37˚C and subsequently replaced with culture medium comprised of Dulbecco's modified Eagle's medium (DMEM; cat. no. C11330500BT; Gibco; Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum (FBS; cat. no. 10100147; Gibco; Thermo Fisher Scientific, Inc.). The mixture was transferred to a 15-ml centrifuge tube. Following centrifugation (200 x g, 4˚C, 5 min), the supernatant was discarded and the sediment was resuspended with DMEM supplemented with 10% FBS and 1% antibiotic solution (penicillin, streptomycin) (cat. no. 15070063; Gibco; Thermo Fisher Scientific, Inc.). The cell suspension was placed into a 25-ml culture flask and incubated at 37˚C and 5% CO₂. The culture medium was changed every 3 days. After 1 week, the ASCs reached a confluency of 90% and the identity was confirmed by immunostaining of S-100 (1:250; cat. no. ab109384; Abcam). After nuclei staining with 5 µg/ml DAPI for 10 mi at room temperature, cells were observed under a fluorescence inverted phase contrast microscope (Olympus Corporation).

The freeze-dried GelMA powder was purchased from MilliporeSigma. The hydrogel was obtained by dissolving 3% (W/V) lyophilized powder and 0.5% (W/V) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) into phosphate-buffered saline (PBS), followed by exposure to 405 nm UV irradiation for 15 sec.

The ASCs were trypsinized, centrifuged (200 x g, 4˚C, 5 min), and then resuspended in 3% (w/v) GelMA solution at the concentration of 1x10⁷/ml. Under the exposure of 405 nm UV irradiation for 15 sec, the cell-seeded GelMA hydrogel was obtained. The hydrogel was then transferred into a six-well plate in which ASC basal medium was added followed by culture at 37˚C with 5% CO₂. Because the gel reflected the light and ASCs were encapsulated in the gels, the cells could be hardly observed under the light microscope. Scanning electron microscopy (SEM, S-3400; Hitachi, Ltd.) was then used to observe the microstructure of the GelMA hydrogel with or without seeded cells. After 1, 3 and 5 days of in vitro culture, the biocompatibility of the GelMA hydrogel with ASCs was evaluated using Calcein-AM/ethidium staining according to the manufacturer's procedure (cat. no. BL3224; Invitrogen; Thermo Fisher Scientific, Inc.).

A total of 80 SD rats were used in the experiments and they were randomly divided into four groups as follows: The sham group (n=20), the SCI group (n=20), the ASC group (n=20) and the GelMA/ASC group (n=20).

Firstly, the healthy adult SD rats were deeply anesthetized with an intraperitoneal injection of pentobarbital (30 mg/kg). With the back shaved and the skin disinfected, a midline incision over the spinous process of ~2 cm was made to expose T9-T11. The paravertebral muscles and the surrounding connective tissue were also cut using micro scissors. The T9-T10 laminectomy was performed. For the latter three groups (SCI, ASC and GelMA/ASC groups), the right lateral hemisection at T10 was performed to create a gap of ~2 mm in length. For the ASC group, the ASC suspension with a cell density of 1x10⁷/ml was injected; for the GelMA/ASCs group, the prepared cell-loaded scaffold was transplanted. The incision was then closed and the rat bladders were manually expressed twice a day until the urinary function recovered. Antibiotics (penicillin, 40,000 µ/kg/day) were also administered to prevent post-operative infection.

The Basso-Beattie-Bresnahan (BBB) scoring system was used to evaluate hindlimb motor function. This is an open-field locomotor evaluation test scoring 21 points, with 0 indicating no movement of hindlimbs and 21 indicating normal levels (23). Prior to the evaluation, the rat was separately placed in an open field with a non-slippery surface and allowed to move freely for 5 min. Two independent observers blinded to the grouping of the rats scored the rat's motor performance according to the BBB scale. The test lasted 4 min and once the rat stopped moving for 1 min, it was placed in the center of the open field again for a re-test.

The inclined plate test was also used for a behavioral assessment. The rat was placed on a plate covered with a 6-mm-thick rubber pad. According to the previous study, the body axis of the rats was perpendicular to the orientation of the plate (24). The plate was then lifted 5˚ every 30 sec and the maximum degree at which the rat could maintain its balance was recorded. Each rat was tested three times and the mean value was the final angle for the individual rat. The behavioral tests were performed from week 1 to week 6 after surgery until all the rats were sacrificed.

At 7 and 42 days after surgery, 3 rats were randomly selected and deeply anesthetized with an intraperitoneal injection of pentobarbital (30 mg/kg). Following heart perfusion with 150 ml pre-cooled PBS containing 4% paraformaldehyde, ~10 mm spinal cord containing the injury site was dissected. It was then post-fixed overnight with 4% paraformaldehyde. The specimen was then embedded in paraffin and sliced into 5-µm-thick sections.

The samples collected at 42 days were stained with hematoxylin and eosin (H&E) to evaluate the lesion volume. Slices were stained with hematoxylin for 10 min and with eosin for 2 min at room temperature. The images were observed under an optical microscope and the volume was measured using ImageJ software (1.49 V; National Institutes of Health).

TUNEL assay kit (cat. no. C1086; Beyotime Institute of Biotechnology) staining was used to evaluate the number of apoptotic cells. The paraffin-embedded sections were stained according to the instructions of the manufacturer. The TUNEL-positive cells with green fluorescence were observed under a fluorescence microscope and six fields of view for each section were randomly selected for subsequent calculation using ImageJ software.

The streptavidin-biotin-peroxidase-complex method was used to detect Bcl-2 expression using immunochemistry. The paraffin-embedded sections were sequentially subjected to standard dewaxing, hydration, antigen retrieval and rinsing, followed by incubation with endogenous peroxidase blockers for 10 min, and sealing in serum for 20 min. After discarding the serum, the primary antibody Bcl-2 (1:250; cat. no. ab196495; Abcam) was added followed by incubation for 24 h at 4˚C. The following day, the sections were rinsed and a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:500; cat. no. ab6721; Abcam) was added, followed by incubation for 30 min at 37˚C. After rinsing thoroughly, the streptomycin-biotin-peroxidase solution was added in a dropwise manner, reacting with the specimen for 20 min followed by diaminobenzidine (DAB) color development. Finally, the nuclei were counterstained with hematoxylin for 2 min at room temperature. Images were observed under an optical microscope and the Bcl-2 positive cells were counted using ImageJ software.

The tissue samples collected at day 7 after surgery were minced using eye scissors on ice and homogenized in 300 µl lysis buffer (cat. no. ST505; Beyotime Institute of Biotechnology) for 30 min. Homogenates were then centrifuged at 15,000 x g for 10 min at 4˚C and the supernatant was collected to quantify the protein concentration using a bicinchoninic acid (BCA) kit (cat. no. A53225; Thermo Fisher Scientific, Inc.) as per the manufacturer's instructions. Subsequently, equal amounts of protein (40 µg per lane) were separated by SDS-PAGE (10% of acrylamide) gel electrophoresis and transferred onto PVDF membrane. The membrane was blocked with 5% bovine serum albumin (BSA; cat. no. 9998; Cell Signaling Technology, Inc.) for 2 h at room temperature and then incubated with primary antibodies at 4˚C overnight. The following day, after three rinses with PBS, the membranes were incubated with the HRP-conjugated goat anti-rabit (cat. no. 7074) and horse anti-mouse (cat. no. 7076) secondary antibodies (1:1,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. The bands were visualized in an enhanced chemiluminescence system (ChemiDox XRS; Bio-Rad Laboratories, Inc.) and images of the target proteins were semi-quantified using ImageJ software. The primary antibodies used were as follows: Anti-Bcl-2 (1:1,000; cat. no. ab196495; Abcam), anti-caspase-3 (1:1,000; cat. no. ab32351; Abcam), anti-p38 (1:1,000; cat. no. 8690; Cell Signaling Technology, Inc.), anti-p-p38 (1:1,000; cat. no. 4511; Cell Signaling Technology, Inc.), anti-ERK1/2 (1:1,000; cat. no. 9194; Cell Signaling Technology, Inc.), anti-p-ERK1/2 (1:1,000; cat. no. 4370; Cell Signaling Technology, Inc.), anti-JNK1/2 (1:1,000; cat. no. 3708; Cell Signaling Technology, Inc.), anti-p-JNK1/2 (1:1,000, cat. no. 9255, Cell Signaling Technology, Inc.), and anti-GAPDH (1:1,000, cat. no. AF0006, Beyotime Institute of Biotechnology).

All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Inc.). The comparison among multiple groups was performed by one-way ANOVA followed by Tukey's post hoc test. Significant differences in BBB locomotion evaluation were determined by the Kruskal-Wallis test with Dunn's multiple comparison. The data are presented as the mean ± standard deviation (SD) or median (IQR). P<0.05 was considered to indicate a statistically significant difference.

At 10 days post-isolation of the ASCs, the ASCs proliferated and covered the T75 culture flask (Fig. 1A). The isolated cells expressed strong red fluorescence of the S100 SC marker after immunostaining (Fig. 1C). A previous study has demonstrated that ASCs exhibit a greater proliferative and adhesive ability than normal SCs (NSCs) (24). Thus, ASCs were used in the following experiments.

The SEM results displayed the 3D porous microstructure of the GelMA hydrogel, which was essential for oxygen and nutrient exchange, as well as for promoting cell survival, proliferation and migration (Fig. 2A). Moreover, it was also found that the ASCs were well encapsulated in the GelMA hydrogel (Fig. 2B).

To further assess the viability of the ASCs encapsulated in the GelMA hydrogel, live/dead staining was performed. The green fluorescence indicated that the majority of encapsulated ASCs were alive and that the cells proliferated from days 1 to 5, as indicated by a gradual increase in green labels (Fig. 2C). Overall, the percentage of alive ASCs was >85% which indicated the favorable biocompatibility of the GelMA hydrogel (Fig. 2D).

Following 48 h of co-culture, the ASC-loaded GelMA hydrogel was transplanted into the hemisection area of the rat spinal cord (Fig. 3A). To determine whether the ASC-loaded GelMA hydrogel could repair the injured spinal cord, H&E staining was performed on the samples collected on day 42 following surgery (Fig. 3B). In the SCI group, H&E staining revealed large cavities and a disordered structure at the injury site. By contrast, the GelMA/ASC groups exhibited an evident decrease in volume and an improvement of tissue integrity (P<0.01). Moreover, compared with the ASC group, the cavities in the GelMA/ASC group were smaller, which demonstrated the improved tissue repair ability of ASCs when encapsulated in the GelMA hydrogel (Fig. 3C).

The ability of the ASC/GelMA hydrogel to repair SCI was also assessed by examining the recovery of hindlimb locomotor function. Thus, the BBB scoring system and inclined plate test were applied to evaluate the motor functional recovery of the rats with SCI from weeks 1 to 6 following surgery. Higher BBB scores or inclined angles represent an improved motor function and vice versa. In the beginning, the rats with SCI exhibited a significant decrease in both BBB scores and inclined angles, suggesting the loss of motor function following SCI. Subsequently, there were overall upward trends in both BBB scores and inclined angles for rats with SCI, suggesting the gradual recovery of their motor function. The GelMA/ASC groups presented significantly higher scores (P<0.05) and inclined angles (P<0.01) than the SCI group from 3 weeks post-injury onward up to the final evaluation (Fig. 4A and B). In particular, the GelMA/ASC group exhibited an improved therapeutic efficacy than single ASC treatment from the second week after surgery to the time of sacrifice.

To investigate cell apoptosis, TUNEL staining and Bcl-2 immunochemistry were performed on the 7th day. For TUNEL staining, compared with the sham group, the number of apoptotic cells in the SCI group was increased (Fig. 5A). The ASC (P<0.01) and GelMA/ASC (P<0.001) groups both exhibited a significantly decreased number of apoptotic cells compared with the SCI group, and GelMA/ASC treatment was found to be more advantageous as compared with single ASC treatment in inhibiting cell apoptosis (P<0.05) (Fig. 5C).

The Bcl-2 positive cells exhibited a brown-stained cytoplasm. In the sham group, a great number of Bcl-2 positive cells appeared, while in the SCI group, only a small number of cells were Bcl-2-immunoreactive (Fig. 5B). In the ASC group, there were more Bcl-2-positive cells than SCI group (P<0.01); however, this number was lower than that in the GelMA/ASC group (P<0.05) (Fig. 5D).

The levels of apoptosis-related proteins, caspase-3 and Bcl-2, were further verified using western blot analysis. The results of quantitative analysis demonstrated that, following SCI, the expression of the pro-apoptotic protein, caspase-3, significantly increased (P<0.01), indicating a higher rate of cell death, while GelMA/ASC transplantation decreased the protein level of caspase-3 (P<0.05). The expression of the anti-apoptotic protein, Bcl-2, was consistent with the results of immunochemistry. Compared with the SCI group, the protein expression of Bcl-2 in the ASC (P<0.05) and GelMA/ASC group (P<0.01) was significantly increased (Fig. 6A and B).

To examine the mechanisms of GelMA/ASCs in inhibiting cell apoptosis following SCI, three important components of the MAPK family were detected, including p38, ERK1/2 and JNK1/2. It was found that the activity of the MAPK family was activated following SCI compared with that of the sham group (p38: P<0.01; ERK1/2 and JNK1/2: P<0.05) (Fig. 6C and D). However, transplantation treatment decreased the phosphorylation level of p38 signaling (ASCs: P<0.05; GelMA/ASCs: P<0.01), while that of ERK1/2 and JNK1/2 signaling were relatively unaltered (Fig. 6C and D). Collectively, the results indicated that GelMA/ASC treatment decreased cell apoptosis following SCI and that these effects may be mediated via the p38 MAPK pathway.