All of the clinical procedures including olfactory mucosa biopsy, bone marrow aspiration, and cell transplantation were performed in Shohada Tajrish Hospital, affiliated with Shahid Beheshti University of Medical Sciences. Inclusion criteria were having a thoracic SCI, complete loss of sensory and motor function below the site of injury (American Spinal Injury Association (ASIA) grade A), at least 6 months post-injury (chronic phase), male or female aged 18 to 70 years, and suffering from no mental disturbance. Exclusion criteria included having severe medical complications or other lesions of the nervous system, spinal stenosis or compression, muscle atrophy, and clinically significant chronic sinusitis or polyps of nasal cavities.
Three patients were enrolled in this trial, all of whom had a chronic, thoracic lesion that resulted either from a road traffic accident or falling from a height. The preoperative neurological evaluation of the patients showed a complete paralysis that was persistent over at least 6 months before the surgery. All three patients had been undergone spinal decompression and fixation surgery at the time of injury. Further assessments to verify eligibility showed no impediment for the mucosal biopsy, bone marrow aspiration, and subsequent cell transplantation (Table 1).
All of the participants went through a regular rehabilitation program started 6 months before the operation and continued until discharge. This scheduled program included physical therapy strategies with a great focus on overground and treadmill locomotor training activities.
Olfactory mucosa biopsy, bone marrow aspiration, and cell isolation
To obtain mucosal biopsies under general anesthesia, the patients were hospitalized. They were placed in a supine position in the operation room. After irrigating and disinfecting the nasal cavities, the biopsy was harvested from the area of superior turbinate using the endoscope. The collected specimens were placed in a cold Hanks’ Balanced Salt Solution (HBSS, Sigma-Aldrich, St. Louis, USA) consisting of 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, Grand Island, USA) and transferred to the cell culture laboratory in a sterile, sealed container. All the next steps were carried out in a class C current good manufacturing practice (cGMP) facility with a class 100 biosafety cabinet under aseptic processing. The isolation of OECs was done according to a protocol described previously (Tabakow et al. 2013). Briefly, the tissue fragments were digested with a 2.4 U/ml dispase II solution (Sigma-Aldrich). After removing the olfactory epithelium, the lamina propria was cut into small pieces and treated with a 5mg/ml collagenase H solution (Sigma-Aldrich), followed by centrifugation and culturing of the cells.
Bone marrow (100 ml) was aspirated from the posterior superior iliac spine of the iliac crest. Based on our previously described method (Oraee-Yazdani et al. 2013), MSC isolation was done using a 1:3 volume of Ficoll solution (1.077 g/L, Sigma-Aldrich). The biphasic prepared sample was centrifuged, and the mononuclear cell layer was separated carefully. After performing three washing steps with HBSS, these cells were also cultured in the appropriate culture conditions.
In-process and final quality control tests
Cell characterization. The characterization of OECs was performed using both S100 and Glial Fibrillary Acidic Protein (GFAP) immunocytochemical staining. The isolated cells were fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 (both Sigma-Aldrich), respectively. The blocking step was done with 10% goat serum (Gibco), followed by incubation with anti-S100 or anti-GFAP (both Santa Cruz Biotechnology, CA, USA) antibody at 4ºc overnight. The samples were then incubated with a suitable horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) for 1 hour and finally exposed to 3, 3'-diaminobenzidine (Sigma-Aldrich) to produce the chromogenic reaction. Hematoxylin (Sigma-Aldrich) was applied for nuclear counter-staining, and cell visualization was carried out through a light microscope.
To confirm the identity of bone marrow-derived cells as MSCs, they were subjected to both flow cytometric analysis of verification markers and differentiation capacity toward adipogenic and osteoblastic lineages (Oraee-Yazdani et al. 2013).
Sterility test and gram stain. The cells were regularly assessed microscopically to confirm their normal growth and the lack of visible contaminations. A direct inoculation sterility test was done every 5 days and also from the final harvested product (Khuu et al. 2006). In brief, a sample was taken from the supernatant of the cultured cell and injected into two microbial culture tubes containing Tryptic Soy Broth and Fluid Thioglycollate Medium (TSB, FTM, both Merck, Darmstadt, Germany). The test tubes were incubated for 14 days at 250c and 350c for TSB and FTM, respectively. Before the transplantation, the standard Gram staining was performed as well on the final cell suspension to verify the absence of contaminating organisms.
Viability assay. A Propidium Iodide (PI) stating was carried out on the final cell mixture before the operation. Following the sample preparation, 1×104 cells/100µl were mixed with a 5-10 µl PI fluorescent agent (Sigma-Aldrich). The sample was then incubated in the dark for 1 min and finally analyzed by FACS Calibur flow cytometer and Flowjo software.
Cytogenetic analysis. The cytogenetic stability of the cultured cells was studied using the standard GTG-banding technique. The cells were delivered to the cytogenetic department of Children's Medical Center and harvested for the conventional karyotype examination.
The cells were separately trypsinized and prepared for transplantation. They were mixed in a ratio of 1:1 of OEC: MSC in 2 ml injectable saline solution (0.9%) at a final concentration of 15×106 cells per ml. Under sterile conditions, the intrathecal injection of the cell mixture was carried out according to our previous study (Oraee-Yazdani et al. 2016). Briefly, the patients were hospitalized and placed in the operating room in a lateral position. After aseptic preparation, the sample was slowly injected into the subarachnoid space of the L4/L5 level through the lumbar puncture using the spinal needle 24 G. The needle was kept in place for one additional minute to avoid leakage.
Pre- and post-operative evaluations
The patients were monitored regularly to record the vital signs and any adverse events based on the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 guideline. The pre-operative Magnetic Resonance Imaging (MRI) scans were carefully analyzed and compared with the ones taken 12 and 24 months post-surgery to track the radiological changes of the spinal cord and its surrounding tissue. To follow the neurological status and functional recovery of the patients, the International Standard of Neurological Classification for Spinal Cord Injury (ISNCSCI) sensory and motor scoring system and the Spinal Cord Independence Measure (SCIM) version III scale were respectively evaluated before and after surgery at 6-month intervals up to 24 months. An electromyography test was carried out when the patients claimed any improvements in their motor activity to confirm that the reported muscle contraction is voluntary.