2-1. Preparation of LNE-loaded gelatin nanofibers
Initially, Gelatin (Type A, Sigma Aldrich) was dissolved in acetic acid (Glacial, Merck) to achieve a final concentration of 28 wt.%. Once a clear solution was obtained, 1 ml of LNE extract (Ethanolic, obtained from Barij, Iran) was thoroughly mixed with 9 ml of the gelatin solution. Subsequently, the LNE-gelatin solution was loaded into a 10 ml syringe connected to an 18-gauge metal needle. Employing a positive high voltage at a magnitude of 18 kV, the polymeric solution underwent electrospinning. The polymer feeding rate was maintained at 0.7-1 ml/hour, the needle-to-collector distance was set at 15-17 cm, and the mandrel's turning rate was adjusted to 500-600 rpm. After the production of scaffolds, they were cross-linked in glutaraldehyde vapor for 1 hour and rinsed three times in distilled water.
2-2 Scanning electron microscopy assay
LNE-gelatin scaffolds and scaffolds lacking LNE underwent a gold coating process lasting 250 seconds before being subjected to SEM imaging at an accelerating voltage of 25 kV.
2-3. Cell viability assay
The viability of ASCs (Pasteur Institute, Iran) cultured on LNE-gelatin and gelatin scaffolds was assessed using Alamar blue assay (Invitrogen, USA). Briefly, scaffolds were sterilized using 70% ethanol for 30 minutes and 15 minutes UV radiation (Each side). Then, ASCs were seeded onto the scaffolds at 10000 cells per cm2 in 50 µl culture media and cultured for 7 days. On days, 2, 4, and 7, 1 ml of Alamar blue solution was mixed with 9 ml of complete culture media and 300 µl of this solution was poured onto the samples and incubated for 3 hours. Finally, the absorbance of samples was read at 570 nm.
2-4. Cell viability assay under oxidative stress
The viability of ASCs cultured on LNE-gelatin and gelatin scaffolds under H2O2-induced oxidative stress was assessed using Alamar blue assay (Invitrogen, USA). Briefly, scaffolds were sterilized using 70% ethanol for 30 minutes and 15 minutes UV radiation (Each side). Then, ASCs were seeded onto the scaffolds at 10000 cells per cm2 in 50 µl culture media and cultured for 48 hours. Then, 1% H2O2 solution was added into each well and cells were incubated for 1 hour. Finally, cell viability assay was performed as described in section 2-3.
2-5. Anti-inflammatory assay
The evaluation of the anti-inflammatory potential of RAW 264.7 macrophage cells was conducted through an ELISA assay. In a nutshell, scaffolds underwent sterilization using 70% ethanol for 30 minutes and received 15 minutes of UV radiation on each side. Following sterilization, macrophages were seeded onto the scaffolds at a density of 40,000 cells per cm² in 50 µl of culture media and were cultured for 36 hours. Subsequently, each well received a supplementation of 1 µg/ml LPS (Sigma), and the cells were incubated for an additional 12 hours. The quantification of pro-inflammatory cytokines, including IL-6 and IL-1β, was carried out using ELISA Assay kits sourced from Abcam, USA.
2-6. DPPH assay
The radical scavenging ability of LNE-gelatin and gelatin scaffolds was evaluated through the implementation of a DPPH assay. Specifically, segments of pure gelatin scaffolds and gelatin scaffolds incorporating LNE were cut into dimensions of 10 × 10 cm². Then, they were immersed in liquid nitrogen and ground into fine powders. Subsequently, various concentrations of these samples were immersed in 3 ml of a 0.1 mM DPPH solution dissolved in methanol, allowing for interaction over a 9-hour period at room temperature.
In this experimental setup, ascorbic acid was employed as the standard control, while DPPH-only samples functioned as the negative control group. To quantify the percentage of DPPH radical scavenging, a spectrophotometric method at 517 nm was utilized. The radical scavenging activity was determined using the formula:
Radical scavenging activity (%) = (Absorbance of Control-Absorbance of control / Absorbance of sample) ×100
2-7. Mechanical strength analysis
The ultimate tensile strength of both gelatin and LNE-gelatin scaffolds was determined using a uniaxial tensile testing device (Santam, Karaj, Iran) with an extension rate set at 1 mm/min, following a methodology inspired by the approach utilized in measuring the tensile strength of manufactured scaffolds. The scaffolds, cut in standardized dimensions, were securely attached to the grips of the testing apparatus. The testing device was configured to apply a uniaxial tensile force at a consistent rate of 1 mm/min until the samples failure.
2-8. Cell adhesion studies
The adhesion of ASCs cultured on LNE-gelatin and gelatin scaffolds was assessed using DAPI staining. Briefly, cells were cultured onto the scaffolds at the density of 40000 cells/cm2 and cultured for 48 hours. Then, the cells were incubated with DAPI dye (Invitrogen, USA) for 15 minutes and then imaged under fluorescent microscope.
2-9. Hemocompatiblity assay
The hemolysis assay for LNE-gelatin and gelatin scaffolds was carried out using rat whole blood that was anticoagulated and subsequently diluted with normal saline. The samples underwent incubation with 200 µl of the blood samples at 37 °C for 60 min, followed by centrifugation at 1500 rpm for 10 min. The resulting supernatant was then analyzed for absorbance at 545 nm using a Multi-Mode Microplate Reader (BioTek Synergy 2). In this assay, the negative control consisted of whole blood diluted in normal saline, while the positive control involved whole blood diluted in deionized water.
2-10. Release assay
Release of LNE from the matrix of electrospun gelatin scaffolds was assessed using UV-visible spectroscopy method. Briefly, 200 mg of LNE-gelatin scaffolds was immersed in 10 ml of PBS and kept at 37 ºC for 3 days. On different time points, 0.5-0.6 ml of the release media was taken and its absorbance was read at 361 nm. The acquired measurements were fitted into the standard curve of LNE in PBS and the amount of release LNE was calculated. Finally, cumulative drug release was reported.
2-11. Preparation of ASCs-seeded constructs for implantation
LNE-gelatin and gelatin scaffolds were sterilized and seeded with ASCs at the density of 40000 cells per cm2 and cultured for 48 hours with DMEM-F12 (Invitrogen, USA) containing 10% FBS (Gibco, USA) and 1% antiobiotics (Gibco, USA). Then, the scaffolds were taken and implanted at the injury site.
2-12. In vivo study
2-12-1. Surgery procedure
In vivo studies This project was approved by the Ethics Review Committee of Guangzhou Ruiye Model Animal Center (Approval No. A202312525). Fifteen male Wistar rats were randomly divided into three groups as follows :1- LNE-gelatin-ASCs, 2- gelatin-ASCs, and negative control (in which the animals received no treatment following injury). The animals were then anesthetized via intraperitoneal injection of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively). Subsequently, the dorsal skin was trimmed, and the surgery region was disinfected using ethanol and povidone iodine. A surgical incision was then executed along the vertebral column, revealing the vertebrae. T-9 laminectomy was performed, involving the incision of the dura matter and the execution of T9 dorsal hemisection. Then LNE-gelatin-ASCs and gelatin-ASCs were implanted at the injury site and fixed in place by suturing. The dura matter, skin, and muscle tissues were subsequently stitched, concluding with the closure of the injury site. The animals were kept for the total duration of 8 weeks.
2-12-2. Behavior factors assessment
The assessment of motor function recovery post hydrogel injection was conducted on weeks 4 and 8 using the Basso, Beattie, and Bresnahan (BBB) experiment, as detailed in a prior study (9). Two impartial observers, unaware of the experimental conditions, examined hind limb movements. In addition, we examined the restoration of sensory function during the fourth and eighth weeks through the Hot Plate Latency (HPL) test. In essence, members of each group were positioned on a hot plate set at 56 ºC, and the duration until the animals exhibited a reaction was recorded. A cut-off time of 12 seconds was established for this experiment.
2-12-3. ELISA assay
On week 8 following the surgery, the animals were sacrificed and their spinal cord tissues were harvested for assessing the tissue expression levels of IL-6, IL-1β, and TNF-α using ELISA assay kits (Abcam, USA).
2-12-4. Histopathological examinations
On week 8th, the animals were sacrificed and the spinal cord tissue at the injury site was assessed for histopathological examinations using Hematoxylin and Eosine (H&E) and Luxal fast blue staining.
2-12-5. Histomorphometry analysis
Volume measurements were acquired through the Cavalieri method employing computer-assisted microscopy. Calculation involved the total volume of intact white matter, unaffected gray matter, lesion volume, and overall cord volume. An observer manually delineated contours using the 4× objective, and the 20× objective aided in defining tissue boundaries. A point-grid with known spacing was randomly superimposed on the live section image, and points within the region of interest were tallied. Subsequently, volume was assessed using the ensuing formula:
V = ∑F × Area × Distance
where, “V” is the total volume, “∑F” is the total number of points hitting region of interest, “Area” is the area associated with each point.
The ultimate post-processing thickness of sections, gauged with the microcator, was applied in the computation of distance. Lesion length was ascertained by determining the distance between the sections at the most rostral and caudal points where no visible lesion or abnormal tissue architecture was observed.
2-13. Statistical analysis
Data was analyzed using Graph pad prism via student’s t-test and one-way ANOVA tests. All experiments were repeated at least three times.