OM-MSCs were obtained from healthy volunteers for scientific research purposes at the Second Affiliated Hospital of Hunan Normal University with ethical approval. All experimental protocols were approved by the Biomedical Research Ethics Committee of Hunan Normal University (approval number 2021–347) and were performed in accordance with the ethical guidelines of the World Medical Association (Declaration of Helsinki). Fresh nasal mucosal tissue was obtained after obtaining signed informed consent from all donors. Three days before surgery, volunteers trimmed their nose hair and used chloramphenicol nasal drops three times a day. As part of the preparation for surgery, the patient’s nasal cavity was thoroughly cleaned and disinfected, and tetracaine was used as a local anesthetic. As soon as the anesthesia is complete, remove about 5 mm³ of superior nasal turbinate tissue and immediately place it in normal saline. Nasal mucosal tissue was cut into 1 mm³ cubes and seeded into 25 cm² culture flasks with standard culture medium consisting of DMEM/F12 (Gibco, Carlsbad, CA, United States) and 10% heat-inactivated fetal bovine serum (FBS, Gibco, Carlsbad, CA, United States). The culture medium was changed every 3 days and maintained at 37°C, 5% CO₂, and 95% humidity. There were a large number of OM-MSCs crawling out of the tissue blocks after 4 weeks of culture. The OM-MSCs were obtained after 5 min of digestion with 0.1% trypsin, the cell is recorded as generation 1. Cells were grown in an incubator at 37°C, 5% CO₂, and 95% humidity, with the medium replaced every 3 days. As soon as the cell confluence reached 80%–90%, the OM-MSCs were digested with 0.25% trypsin-EDTA (Gibco, Carlsbad, CA, United States) for 30 s, and digestion was terminated with complete medium containing fetal bovine serum. A ratio of 1:4 was used to subculture the cells, and high-purity OM-MSCs were obtained following the fourth generation.

Preparation of the fourth generation OM-MSCs into cell suspension, add 100ul of cell suspension to each tube, and then add 2ul of CD34, CD45, CD73, CD90, CD105 antibodies (Invitrogen, Waltham, MA, United States) to stain the cells, add anti-IgG-PE (Invitrogen, Waltham, MA, United States) to the negative control tube, and incubate in a 4°C refrigerator away from light for 30 min. Wash the cells twice with PBS, and then put the samples into the flow cytometer for detection. It was found that the purity of the OM-MSCs was greater than 98%.

Inoculate 5 × 10⁴ cells into the wells of a 6-well plate with added culture medium. After the cells have attached, replace media with pre-warmed complete adipogenesis differentiation medium (Gibco, Carlsbad, CA, United States) and continue incubation. Each well of the six-well plate was filled with 2 mL of differentiation medium, which was replaced every 3 days according to the manufacturer’s instructions. Three weeks after cultivation, remove the culture medium and clean with PBS three times. 2 mL of 4% paraformaldehyde solution should be added to each well of the six-well plate, and the solution should be fixed for 10 min, followed by three washes with PBS. For staining, add 2 mL of Oil Red O dye working solution to each well and incubate for 30 min at room temperature. After three washes with PBS to remove excess dye, the specimens were observed under a light microscope.

Inoculate 5 × 10⁴ cells into the wells of a 6-well plate with added culture medium. After the cells have attached, replace media with pre-warmed complete osteogenesis differentiation medium (Gibco, Carlsbad, CA, United States) and continue incubation. Each well of the six-well plate was filled with 2 mL of differentiation medium, which was replaced every 3 days according to the manufacturer’s instructions. 21 day after cultivation, remove the culture medium and clean with PBS three times. 2 mL of 4% paraformaldehyde solution should be added to each well of the six-well plate, and the solution should be fixed for 10 min, followed by three washes with PBS. For staining, add 2 mL of Alizarin Red Staining Solution to each well and incubate for 10 min at room temperature. After three washes with PBS to remove excess dye, the specimens were observed under a light microscope.

15 × 15 × 5 mm³ gelatin sponge was washed 3 times with PBS in advance and immersed in 0.75 mg/L PLL solution (Beyotime, Shanghai, China) at room temperature overnight. After washing 3 times with PBS, the gelatin sponge was placed in the center of a six-well plate, and 1 mL of complete culture medium containing 1 × 10⁶ OM-MSCs was evenly inoculated on the surface of the gelatin sponge and incubated in a humidified 5% CO₂ at 37°C for 2 h. Subsequently, 2 mL of complete culture medium was added to the six-well plate, and the morphological changes of the gelatin sponge were photographed with a mobile phone during the entire experiment.

When OM-MSCs were cultured in gelatin sponge for 1, 3, 5, 10, 15, 20, 25 and 30 days, the cell supernatant was aspirated, CCK-8 reagent (Beyotime, Shanghai, China) and complete culture medium were mixed in a ratio of 1: 9, and 2 mL was added to a 6-well plate. After gently shaking, the plate was incubated in a humid 5% CO₂ at 37°C for 0.5 h, and then the supernatant was aspirated and transferred to 100 μL in a 96-well plate, with 6 replicate wells in each group; the 96-well plate was placed in an reader to detect the absorbance value at an excitation wavelength of 450 nm, and the cell viability was calculated.

The levels of lactate dehydrogenase (LDH), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in spinal cord tissues from Sprague-Dawley (SD) rats were quantified using an ELISA kit (4A Biotech, Suzhou, China). After incubation with a working solution, samples were exposed to a substrate solution at 37°C for 0.5 h. After adding the termination solution, the absorbance was measured using a microplate reader (Thermo Fisher, United States).

Discard the supernatant of the corresponding days of gelatin sponge and wash it twice with PBS. Use Calcein-AM/PI live/dead cell double staining kit (Solarbio, Beijing, China) to stain the cells. Follow the instructions and incubate in a cell culture incubator. After incubation, aspirate the staining solution, add PBS, and observe and photograph the live and dead conditions of OM-MSCs in the gelatin sponge under a fluorescence microscope.

All animal experiments were performed in accordance with the guidelines approved by the Biomedical Research Ethics Committee of Hunan Normal University (approval number 2021–347). Female Sprague Dawley rats (200–220 g), 8 weeks old, were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). All rats were housed in a temperature-controlled room (22°C ± 1°C) with 12 h light/12 h dark cycle. Forty female SD rats were randomly divided into sham operation group, SCI group, GS group, and GS + OM-MSCs group, with 10 rats in each group. We established a rat model of completely transected SCI, with a 28-day observation period after modeling. An intraperitoneal injection of sodium pentobarbital (50 mg/kg) anesthetized the rats, and the target T10 vertebra was located through the ribs, an incision was made with a scalpel, and the surrounding tissue of the vertebra was bluntly separated. The muscles were cut and separated layer by layer to expose the T10 lamina and spinous process. The spinous process and lamina were only bitten off with bone rongeurs to expose the spinal cord, and the remaining bone structures and intact dura mater were preserved. To establish a completely transected spinal cord injury, the SCI group, GS group, and GS + OM-MSCs group used ophthalmic scissors to remove 3 mm columnar spinal cord tissue. The Sham group was left untreated except for laminectomy. Both the GS and GS + OM-MSCs groups had the spinal cord defects filled with gelatin sponges, followed by layering sutured muscles, fascia, and skin, and disinfected external skin. Postoperatively, the animals were resuscitated in an incubator and provided with adequate food and water. Penicillin (2 × 10⁶ U/kg/day) was administered for 3 days to prevent infection, and the bladder was massaged twice daily until the bladder function was restored. Record the daily survival status of each group and plot the overall survival curve.

After surgery, functional recovery following SCI was evaluated utilizing the Basso, Beattie, and Bresnahan (BBB) assessment and footprinting. Two blinded investigators evaluated sensory and motor functions on postoperative days 0, 7, 14, 21, and 28 in an unconfined setting. The assessment scale, spanning from 0 (representing paralysis of the whole body) to 21 (representing full functional recovery), was recorded independently by each investigator, and the composite score was derived by computing the mean of the evaluations. From each group, five surviving rats were randomly selected and marked with blue and red ink on their forelimbs and hindlimbs, then allowed to walk through a path lined with white paper. Two impartial observers independently examined the footprints without knowing the parameters of the experiment.

Immunohistochemistry was used to evaluate the day 28 spinal cord. On the day of sacrifice, rats were deeply anesthetized and perfused intracardially with PBS followed by 4% paraformaldehyde (PFA). The spinal cord was then removed, fixed in 4% PFA for 24 h, embedded in paraffin, and cut into serial longitudinal sections with a thickness of 10 μm using a cryostat (LEICA RM2125 RTS, Leica Biosystems, Nussloch, Germany).

Hematoxylin-eosin (H&E) staining for evaluation of spinal cord tissue structure. The sections of brain sections were deparaffinized with graded ethanol together with xylene, and 4 μm sections were prepared for HE staining.

Nissl staining for assessment of neuronal damage. Cresyl violet (Beyotime, Shanghai, China) was used to stain brain sections for 30 min at room temperature. Next, brain slices were washed with distilled water, treated with 95% ethanol for 30 s, covered with 50% glycerol, and dried.

Immunofluorescence staining was performed on day 28 specimens to evaluate pyroptosis. Briefly, Spinal cord sections were incubated overnight at 4°C with NLRP3 (Abcam, Fremont, CA, United States) and Caspase-1 (Abcam, Fremont, CA, United States) and then with Alexa Fluor 594 goat anti-mouse (Beyotime, Shanghai, China). Mounting medium containing DAPI (Beyotime, Shanghai, China) was used to visualize nuclei.

The tissues were processed for Western blot analysis. We lysed the samples using RIPA lysis buffer (Beyotime, Shanghai, China). We followed the instructions according to the instructions provided by the manufacturer. We determined the protein concentration using a BCA protein assay kit (Beyotime, Shanghai, China). Each group of proteins from the adjusted samples were loaded onto the gel and the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were then blocked with 5% skim milk for 1 h and incubated with primary antibodies overnight at 4°C. The antibodies used in this study included NLRP3 (dilution 1:1,000, 27458-1-AP, Proteintech, China), Caspase-1 (dilution 1:1,000, ab207802, Abcam, United States), GSDMD (dilution 1:2000, ab209845, Abcam, United States), β-actin (dilution 1:5,000, 10663-1-AP, Proteintech, China), IL-1β (dilution 1:1,000, bs-0812R, Bioss, United States) and IL-18 (dilution 1:2000, 27458-1-AP, Proteintech, China). After three washes with TBST, the membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies. Finally, imaging was achieved using ECL chemiluminescence substrate (BL520B; Biosharp, China), and the intensity of the bands was quantified using ImageJ software.

The collected SD rat spinal cord or Gelatin sponges were fixed overnight at 4 °C with 2.5% glutaraldehyde, followed by washing in cacodylate buffer and fixation with 1% osmium tetroxide. After another round of washing in cacodylate buffer, the samples were embedded in a double-staining solution containing 5% uranyl acetate and lead citrate before being sectioned using an ultramicrotome for examination under an electron microscope (HT7700, Hitachi, Tokyo, Japan).

OM-MSCs were labeled with GFP (Genechem, Shanghai, China) in advance. SCI rats were imaged using the IVIS Lumina II imaging system 28 days after surgery. Analysis was performed based on regions of interest (ROI).

We present the data as a mean ± standard deviation (SD). Student’s t-tests,or oneway analysis of variance (ANOVA) with the Bonferroni corrections for post hoc t-test, or two-sided ANOVAs with Bonferroni corrections for post hoc t-tests were conducted to estimate differences between groups. GraphPad Prism 10 Software was used for the statistical analysis (La Jolla, CA, United States). Each experiment was replicated at least three times. P-values less than 0.05 were considered significant.

The OM-MSCs were isolated and purified according to previously described protocols (Zhuo, 2024), and their cell surface markers and differentiation capacity were characterized (Supplementary Figure S1). 1 × 10⁶ OM-MSCs were seeded in a 15 × 15 × 5 mm³ gelatin sponge, and their morphology was observed. As the culture time increased, the gelatin sponge size decreased. After 10 days of culture, the edges gradually became rounded and the transparency became higher and higher. Gelatin sponges showed jelly-like properties after 20 days of culture. Early in the culture process, OM-MSCs were sparsely distributed in the gelatin sponge (Figure 2A). After 15 days of culture, the OM-MSCs were evenly distributed throughout the material, and the number of OM-MSCs increased (Figure 2B). After 30 days of culture, the distribution of OM-MSCs in the gelatin sponge significantly changed, and the number of cells distributed on the surface was significantly greater than that in the center (Supplementary Figure S2A). It is possible that this is the reason why the center of the gelatin sponge collapsed after 30 days of culture (Figure 1A).

We performed live-dead staining on OM-MSCs in gelatin sponges within 30 days of culture to determine the optimal culture time for OM-MSCs (Figure 1A). On the gelatin sponge, OM-MSC viability declined after 10 days, and a large number of cells died after 15 days (Figure 1B). Under the same culture conditions, conventionally cultured OM-MSCs and OM-MSCs loaded on gelatin sponge were compared for cell viability. A CCK-8 study showed that conventionally cultured OM-MSCs slowed down after 5 days, reaching peak viability on day 10. In contrast, OM-MSCs loaded on gelatin sponge reached their peak viability on day 15, showing a better cell proliferation effect than conventional culture (Figure 1C). We chose 15 days as the duration for culturing OM-MSCs in the gelatin sponge since cells cultured for 10–15 days have a strong proliferation ability and have a relatively small number of dead cells. Compared with conventional 2D culture, gelatin sponge can provide OM-MSCs with an environment that enhances cell viability, which is one of its advantages.

In comparison with the 3 days culture group, OM-MSCs grew in the gaps of the gelatin sponge after 15 days of culture (Figures 2A,B). Based on scanning electron microscopy (SEM) observations (Figure 2C), the gelatin sponges without OM-MSC growth had a loose and porous structure with pores between 50 and 300 μm, which enabled mesenchymal stem cells to gro. The gelatin sponge was filled with OM-MSCs after 15 days of growth (Figure 2D), indicating good biocompatibility. OM-MSCs grown for 30 days on gelatin sponge showed no significant increase in cell number and many fragmented nuclei, indicating cell death after HE staining and SEM (Supplementary Figure S2A). In order to further investigate the biocompatibility of gelatin sponge with OM-MSCs, we detected markers of extracellular matrix. OM-MSCs, laminin, fibronectin and cell nuclei were labelled after 15 days of gelatin sponge culture and laminin and fibronectin expression increased significantly (Figure 2E). A biocompatible combination of OM-MSC and gelatin sponge is demonstrated here.

It is common for incomplete spinal cord injuries to result in better recovery of motor function, but this is not necessarily a result of the spinal cord tissue healing on the injured side (Friedli et al., 2015). Therefore, we performed a complete spinal cord injury model on the T10 segment of 40 SD female rats. We performed local transplantation of cell-free gelatin sponges or OM-MSC-loaded gelatin sponges immediately after modeling (Figure 3A). After modeling, motor function evaluations and pathological analyses were performed on the Sham group (only the spinal cord was exposed), the SCI group (the spinal cord was completely cut), the GS group (the spinal cord was completely cut and gelatin sponge was transplanted), and the GS + OM-MSCs group (the spinal cord was completely cut and gelatin sponge loaded with OM-MSCs was transplanted).

As inflammation often occurs in the early stages of spinal cord injury and affects repair later, we set a 4-week observation period. 28 days after surgery, the hind limb performance of the rats in each group was as follows: The rats treated with GS were able to walk using plantar, whereas SCI group rats were not (Figure 3B). In the early period after surgery, particularly in the SCI group, rats had a relatively high mortality rate, as shown by the survival curve (Figure 3C). On the day of SCI modeling and 1–4 weeks after modeling, SD rats were scored for Basso–Beattie–Bresnahan (BBB) scores to determine the long-term effect of OM-MSCs on motor recovery (Figure 3D). The pre-modeling score of each group was 21 points, and on the day of SCI modeling, the total score of the rats who underwent complete spinal cord injury modeling was 0 points, and the average BBB score gradually increased in the following 1–4 weeks 14 days after surgery, the BBB scores of rats in the GS + OM-MSCs group were higher than those in the SCI group. However, there was no significant difference between the GS group and the SCI group (Figure 3E). 28 days after surgery, the BBB score showed that the GS + OM-MSCs group was significantly higher than the GS group. However, there was no significant difference between the GS group and the SCI group (Figure 3F). The results showed that transplantation of gelatin sponges was helpful for functional recovery in rats with spinal cord injury. The repair rate was significantly reduced with time, but gelatin sponges loaded with OM-MSCs continued to have functional repair effects.

28 days after surgery, we performed gait analysis in each group of rats. We smeared the rat forefoot with blue ink and the rat forefoot with red ink, then passed on a runway with a length of 600 mm and a width of 100 mm (Figure 3G). As the gait diagram shows, the hindlimbs of the SCI group dragged mostly, whereas the GS and GS + OM-MSC groups dragged less, and the footprints of the GS + OM-MSC group were clearer. According to the results of gait analysis, we counted the relative area of the hind limbs and forefoot of the rats in each group, and found that the relative areas of the hind limbs in the GS + OM-MSCs group and the GS group were lower than those in the SCI group, and the contact area of the hind limbs in the GS + OM-MSCs group decreased the most significantly (Figure 3H). In gait analysis, the average stride length was significantly greater in the GS group and the GS + OM-MSCs group than in the SCI group, but the difference in stride length between the GS group and the GS + OM-MSCs group was not significant, probably due to similar degrees of recovery in the large joints of the hind limbs (Figure 3I). SCI rats can walk better after receiving gelatin sponge transplants, and OM-MSCs can enhance this effect.

According to histological examination of the SCI group, an infiltrating cavity appeared in the middle of the lesion, with inflammatory cells infiltrating it, and evident vacuoles on both sides of the lesion, suggesting that the inflammation is significant. Comparatively, spinal cord tissue lesions were milder in the GS group and GS + OM-MSCs group (Figure 4A). Probably, the spinal cord tissue material supports tissue regeneration after spinal cord injury, which promotes nerve cell growth. Interestingly, in the spinal cord section, we found unabsorbed gelatin sponge material encased in the spinal cord tissue (Figure 4B). GS + OM-MSCs had similar tissue structure to GS, but there was no unabsorbed gelatin sponge material in the sections. Since OM-MSCs were cultured with gelatin sponge for 15 days in vitro, the cells and sponge tissue have merged, and they are more easily absorbed after being transplanted into rat spinal cord tissue, which demonstrates gelatin sponge’s tissue compatibility.

Nissl staining revealed few Nissl bodies in the spinal cords of rats with spinal cord injuries, indicating severe nerve damage (Figure 4C). In the GS group, only a small number of neuronal cell bodies were observed; in the GS + OM-MSCs group, neuronal cell bodies increased in number and size, suggesting that the transplanted gelatin sponge and OM-MSCs protected neurons.

Through ELISA detection, we further analyzed the levels of related inflammatory factors in the tissues. The levels of LDH, IL-1β, and TNF-α were highest in the SCI group, while these inflammatory markers were further reduced in the GS group and the GS + OM-MSCs group (Figures 4D–F). Based on the above, we found that the transplantation of gelatin sponges could alleviate the neuroinflammation of a part of the spinal cord tissue. The effect of gelatin sponges loaded with OM-MSC was more obvious.

According to histology and related detection of inflammatory factors, the local lesions of spinal cord injury rats are in a state of inflammation for a long time, leading to the death of a large number of neurons, which may be an important reason affecting their functional recovery (DiSabato et al., 2016; Orr and Gensel, 2018; Pang et al., 2021). The cellular structure of neurons and microglia in spinal cord tissue from SCI rats was examined using TEM. There were enlarged neuronal cell bodies, multiple discontinuities in cell membranes, vacuoles in the cytoplasm, enlarged mitochondria, and swollen mitochondrial cristae, all suggesting that damage had been done to organelles such as mitochondria, endoplasmic reticulum, and Golgi apparatus (Figure 5A). The SCI group showed multiple holes in microglia, disordered cytoplasm, and swollen mitochondria (Supplementary Figure S3). The typical cell structural features suggest that the cells are undergoing pyroptosis. IL-1β and IL-18 can also be released due to cell pyroptosis, increasing the inflammatory response. Therefore, we speculate that the inflammatory response in spinal cord injury is related to pyroptosis.

In pyroptosis, proteins such as IL-1β, IL-18, GSDMD, Caspase-1, and NLRP3 cooperate to enhance inflammatory responses and death of cells. As a core component of the inflammasome, NLRP3 detects intracellular danger signals and initiates the pyroptosis signaling cascade. WB detection showed that the NLRP3 protein level in the SCI group was significantly increased (Figures 5B,J). However, the protein content of the pyroptosis-related NLRP3 inflammasome was significantly reduced in the GS group and the GS + OM-MSCs group.

The NLRP3 inflammasome can further activate Pro-Caspase-1 to become active Cleavage-Caspase-1 (Swanson et al., 2019). As shown in the test results (Figures 5B,G,H,I), the level of Pro-Caspase-1 is increased in the SCI group, whereas it is not significantly reduced in the GS group, only in the GS + OM-MSCs group. Further, the GS group and the GS + OM-MSCs group showed significantly reduced levels of Cleavage-Caspase-1 (22 kDa) and Cleavage-Caspase-1 (20 kDa).

Gasdermin D (GSDMD) is a pyroptosis execution protein (Shi et al., 2017). Caspase-1 or Caspase-4/5/11 cleave GSDMD to generate active N-terminal fragments in response to intracellular inflammation. The N-terminal fragment of GSDMD can insert into the cell membrane and form pore-forming, which damages the integrity of the cell membrane, causing cell contents leakage and triggering pyroptosis. The test results showed that the full-length GSDMD in the SCI group was significantly increased, but the levels in the GS group and GS + OM-MSCs group were not substantially reduced. On the contrary, the level of active GSDMD-N was significantly reduced in the GS group and the GS + OM-MSCs group (Figures 5B,E,F).

Pyroptosis induces an inflammation dependent on IL-1β and IL-18 (Chen et al., 2023). Through the membrane pores formed by GSDMD, IL-1β and IL-18 can be rapidly released into the extracellular space, further prolonging the inflammatory response. From the test results, it can be seen that the levels of IL-1β and IL-18 in the SCI group were significantly increased, and the levels in the GS group were not significantly reduced, but the levels in the GS + OM-MSCs group were considerably lower than those in the SCI group (Figures 5B–D).

The activation of GSDMD causes the degradation of membranous organelles such as mitochondria in pyroptotic neurons and irreversible damage to the plasma membrane around the ischemic region (Sborgi et al., 2016). As compared to the SCI group, GS and GS + OM-MSCs significantly reduced the rupture of the membrane, cytoplasmic disorder, and mitochondrial swelling in neurons. The GS group and GS + OM-MSCs group also showed improved cell structure and organelle morphology than the SCI group. Moreover, mitochondrial swelling and cytoplasmic disorder were reduced further in the GS + OM-MSC group, and more normal mitochondria and organelles were retained (Figure 5A).

A study of immunohistofluorescence in spinal cord tissue revealed increased expression of Caspase-1 and NLRP3 in the SCI group, while after gelatin sponge transplantation, pyroptosis-related markers were downregulated, especially in the GS + OM-MSCs group, which was more effective at inhibiting pyroptosis (Figures 5K–M). According to the results of the above tests, gelatin sponges and OM-MSC transplantation have been shown to inhibit pyroptosis in rats following spinal cord injury.

28 days after the OM-MSCs loaded with gelatin sponge were transplanted into SCI rats, HE staining analysis was performed on multiple organs of the rats. The results showed that the rats’ brain, heart, spleen, lung, liver, and kidney tissue structures were normal 28 days after transplantation (Figures 6A–F). In order to observe whether OM-MSCs can survive in vivo, we used green fluorescent protein to mark OM-MSCs in advance, and used small animal in vivo imaging to observe the fluorescence signal of OM-MSCs at the surgical site of the rats 28 days after transplantation (Figures 6G,H). GS + OM-MSCs group displayed obvious GFP fluorescence signals at the surgical site. In short, the above results show that OM-MSCs can survive in rats, and gelatin sponge and OM-MSCs have advantages in biocompatibility.