rADSCs were obtained according to the previous protocols (11,17,18). Briefly, the adipose tissues were dissected from subcutaneous fat in the inguinal region of Sprague-Dawley male rats. The tissues were washed three times with phosphate-buffered saline (PBS) and minced with scissors. Then, tissues were digested in PBS containing 2 mg/ml collagenase type II (Sigma-Aldrich, St. Louis, MO) for 40 min at 37°C with constant agitation. Then, tissues was mixed with an equal volume of Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY), passed through a 40-µm Nylon cell strainer (BD Falcon, San Jose, CA) and centrifuged for 10 min at 1,500 g. Afterward, floating adipocytes were removed from the stromal vascular fraction. Finally, the obtained cells were seeded on cell culture flasks (Thermo Fisher Scientific, Waltham, MA) supplemented with α-modified Eagle's medium (Gibco, Grand Island, NY), 10% FBS, and 1% penicillin-streptomycin (Gibco, Grand Island, NY). They were incubated at 37°C in a humidified atmosphere under 5% CO₂. The culture medium was replaced every 2–3 days. Cells were trypsinized with 0.25% trypsin-EDTA (Gibco, Grand Island, NY) and expanded in new flasks when they reached 70–80% confluence. Cells used for transplantation in ASCs were from passages 3 to 6.

For osteogenic differentiation, rADSCs at passage 3 were inoculated at 4×10³ cells/cm² and cultured for 2 weeks in osteogenic induction medium [MEM containing 10% FBS, 100 µg/ml ascorbate, 0.1 µM dexamethasone and 10 mM β-glycerophosphate (All from Cyagen Biosciences, Suzhou, Jiangsu, China)]. rADSCs were then fixed with 4% paraformaldehyde (PFA, Boster Biosciences, Beijing, China) for 20 min, washed with PBS for 3 times, afterward, incubated with 1 mg/ml Alizarin Red (Sigma-Aldrich, St. Louis, MO) solution for 30 min to stain for calcium deposition. For adipogenic differentiation, rADSCs at passage 3 were cultured for 2 weeks in adipogenic induction medium [high glucose-DMEM containing 10% FBS, 1 µM dexamethasone, 0.5 mM methyl-isobutylxanthine, 10 µg/ml insulin, and 100 µM indomethacin (All from Cyagen Biosciences, Suzhou, Jiangsu, China)]. Then, cells were fixed with 4% PFA for 20 min, washed with PBS for 3 times, afterward, immersed in 0.3% Oil Red O solution (Sigma-Aldrich, St. Louis, MO) in 60% isopropanol for 30 min to assess lipid droplet formation.

Surface antigens of the undifferentiated rADSCs were identified by flow cytometry using anti-CD29 (1:200, Biolegend, San Diego, CA), CD90 (1:100, Biolegend, San Diego, CA), CD44 (1:400, Abcam, Cambridge, UK) and CD45 (1:200, Biolegend, San Diego, CA) antibodies. rADSCs were resuspended in buffer containing PBS supplemented with 1% bovine serum albumin (BSA, Gibco, Grand Island, NY) to reach a concentration of 1×10⁷ cells/ml after trypsinizing with 0.25% trypsin-EDTA, and incubated with fluorescence-conjugated primary antibodies for 30 min on ice. Flow cytometry analysis was performed on the BD Accuri™ C6 Flow Cytometer System (BD Biosciences, NJ, USA) and data were analyzed using Cflow Plus software (Becton-Dickinson, NJ, USA).

The rat ASCs were harvested as previously described according to standard procedures developed by our colleagues (6,7). In short, SD rats were anesthetized with intraperitoneal injection of sodium pentobarbital (45 mg/kg body weight). Thoracic spinal cord samples (~20 mm) were obtained and then rinsed in PBS to remove blood. Obtained tissues were incubated in distilled water (changed per 2 h) at room temperature for 6 h. Then, samples were serially immersed in 1% Triton X-100 solution (3 h), PBS (3 h), 1% sodium deoxycholate solution (3 h), and PBS (3 h), respectively. Meanwhile, specimens were oscillated at 100 rpm throughout the immersion process. Afterward, samples were lyophilized to obtain the acellular spinal cord scaffolds after serial immersions were repeated once. The lyophilized scaffolds were incubated in genipin and glutaraldehyde solutions (both 5 mg/ml in 0.01 M PBS, pH 7.4), respectively, for chemical crosslinking for 24 h at 37°C. All specimens were immediately freeze-dried for 24 h in freeze drier and sterilized by irradiation (16 kGy) with Cobalt-60 gamma rays before engraftment. Before transplantation, rADSCs were cultured in 10 µM 5-bromodeoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO) for 48 h. Subsequently, they were injected into ASCs (0.2×0.1 cm) at a concentration of 5×10⁶ cells/ml. The engraftments were then incubated at 37°C in a humidified atmosphere under 5% CO₂ in rADSCs culture medium for 2–3 days, stained with hematoxylin and eosin (HE), and examined by light microscopy. The ultrastructure of the engraftment were visualized by scanning electron microscopy (SEM, S-3400 N, Hitachi, Japan) (7).

All animal experiments were approved and supervised by the ethics committee of The Third Military Medical University according to international standards for animal welfare. Adult male SD rats (body weight, 200–250 g) were purchased from animal center of The Third Military Medical University and anesthetized with intraperitoneal injection of sodium pentobarbital (45 mg/kg body weight). Thoracic spinal cord segments were exposed after laminectomy at the T9-10 level under aseptic condition. Two right-sided hemisections of spinal cord were performed by microdissection scissor between T9 and T10 level under surgical microscope. A 2 mm-width gap was created with a 22-gauge syringe needle (8). Then, muscle layers and skin were sutured separately. To prevent infection, rats were subcutaneously injected with ampicillin (100 mg/kg) and gentamicin (12 mg/kg) following 3 days after surgery (once a day). Their bladders were emptied twice daily until they regained bladder control. Rats were randomly divided into 3 groups after hemisected SCI: (1) without any intervention (SCI only, n=15), (2) with ASC scaffolds implantation (ASC only, n=15), (3) with ASC scaffolds seeded with rADSCs transplantation (ASC + ADSCs, n=15). Behavioral tests (including BBB locomotor rating scale, hypersensitivity to mechanical and thermal stimulation) were assessed weekly before and after surgery. Histological examinations and immunohistochemistry were performed on all animals. BDA anterograde tracing were used to detect nerve fibers in injuried rat spinal cord on day 14 post-SCI.

For bsehavioral tests, two independent investigators blinded to the experimental design separately assessed and scored behavioral recovery. The locomotor recovery after SCI was evaluated in view of the BBB score as previously described (19) on day 1, 7, 14, 21 and 28 post-SCI.

Rats were anesthetized and placed on a stereotaxic frame and biotinylated dextran amines (BDA, Invitrogen, Breda, Netherlands) was injected in view of previous study before conducting surgery at spinal cord (20).

Scaffolds and tissue sections were fixed in 4% paraformaldehyde, paraffin-embedded, sliced, and stained with hematoxylin and eosin (H&E, Beyotime C0105, Beijing, China) according to the manufacture's instructions (21). The stained samples were visualized using a light microscopy (Leica, Wetzlar, Germany).

The histological analysis was employed according to standard procedures (8) and the antibodies were as follows: Mouse anti-Brdu (1:200, Abcam, Cambridge, UK), rabbit anti-glial fibrillary acidic protein (GFAP, 1:300, Abcam, Cambridge, UK).

For Brdu staining, sections were firstly incubated in 2 M HCL for 30 min, then neutralized with 0.1 M sodium borate buffer (pH 8.5) for 30 min at room temperature for DNA hydrolysis (22). Slices were visualized by a light microscopy (Leica, Wetzlar, Germany).

For BDA tracing, three sagittal slices were selected from each group and rinsed with PBS, then treated with avidin-Cy3 containing 0.25% TritonX-100 in PBS at room temperature for 2 h and rinsed in PBS for three times for analysis.

All sections with immunofluorescence were examined by a confocal microscope (Carl Zeiss, LSM780, Weimar, Germany) and analyzed using Zen 2011 software (Carl Zeiss, Weimar, Germany).

Data were represented as the mean ± SEM and data analysis were conducted using SPSS V18.0 (SPSS Inc, Chicago, IL). Comparisons among multiple sets were performed using One-way ANOVA followed by Tukey's post hoc test and a P<0.05 was considered as significance.

rADSCs expressed cell surface antigen CD29, CD44 and CD90 after 3 passages, but negative for CD45 by flow cytometry analysis (Fig. 1A). To assess the differentiation potential of cultured cells, they were incubated in induction media for 14 day to evaluate their differentition into osteocytes and adipocytes, respectively. The results indicated that cultured cells held the capability of osteogenesis (Fig. 1B) and adipogenesis (Fig. 1C). To test the biocompatibility of ASCs, rADSCs pre-cultured in 10 µM BrdU were seeded into ASCs, which appeared to be white color and became soft and flexible two days latter. Brdu⁺ cells were interspersed in the network of ASCs before transplantation (Fig. 1D). Meanwhile, rADSCs with thin fibers forming uniform mesh pores were observed using SEM (Fig. 1E). In short, these results indicated rADSCs were able to survive and exert biological function in ASCs, which were safe and biocompatible, according to our established procedures.

To explore the functional benefits with ASCs seeded with rADSCs, BBB scores, hypersensitivity to mechanical and thermal stimulation assays were performed on day 1, 7, 14, 21 and 28 post-SCI in three groups: SCI only, ASC only and ASC + ADSCs. The data represented that rats in ASC + ADSCs group showed the best improvement in locomotor recovery. Meanwhile, rats in ASC only group represented better than those in SCI only group from day 14 after SCI (Fig. 2A). Moreover, rats in ASC + ADSCs group depicted the same trend as locomotor renovation in the other two functional tests of hypersensitivity to mechanical and thermal stimulation assays (Fig. 2B and C). Together, the functional tests data demonstrated that implantation of ASCs seeded with rADSCs was able to enhance functional recovery after SCI in rats.

To illuminate the reason why functional recovery improved greatly in rats receiving engraftment of ASCs seeded with rADSCs, H&E staining was used to observe histopathological changes on day 14 post-SCI. The images represented that rats in ASC + ADSCs group showed significant diminution and less atrophy of the injured site than that in the ASConly and SCI only groups (Fig. 3B). Meanwhile, the semi-quantitative data revealed the lesion area in ASC + ADSCs group decreased most than that in the ASC only and SCI only groups. Furthermore, ASC only group indicated better improvement than that in the SCI only group (Fig. 3C). In addition, a certain number of Brdu⁺ rADSCs survived in the grafted ASCs and distributed over the host spinal cord in ASC + ADSCs group on day 14 after SCI (Fig. 3D), which was in line with previous study in hUCB-MSCs transplantation (8).

Our previous research implied that ASCs manufactured by our colleagues might potentially support axon regeneration after nervous system injury (6). Here, we conducted BDA anterograde tracing with immunofluorescence staining showing that transplantation of ASCs seeded with rADSCs held the best improvement potential in harnessing axon regeneration to bridge lesions in ASC + ADSCs group than that in the ASC only and SCI only groups. BDA positive area in ASC only group was larger than that in the SCI only group (Fig. 4A). Meanwhile, the semi-quantitative data indicated similar tendency observed from Fig. 4A (Fig. 4B). Moreover, we performed GFAP staining to assess reactive gliosis in lesions. The results indicated that GFAP⁺ area was much smaller in ASC + ADSCs group than that in the ASC only and SCI only groups (Fig. 4C and D). Furthmore, there was no obvious difference between ASC only group and SCI only group (Fig. 4C and D). Taken together, our data certified that ASCs manufactured by our colleagues could support axon regeneration and implantation with ADSCs enhanced this effectiveness after SCI. In addtion, it reduced reactive gliosis, and might finally decreased glial scar formation when transplantation ASCs seeded with ADSCs.