Gene therapy of spinal cord injury using gene-modified Bone Marrow Stromal Cells with Fibromodulin expressing adenoviral vector in a rat SCI model

Gene therapy of spinal cord injury using gene-modified Bone Marrow Stromal Cells with Fibromodulin expressing adenoviral vector in a rat SCI model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gene therapy of spinal cord injury using gene-modified Bone Marrow Stromal Cells with Fibromodulin expressing adenoviral vector in a rat SCI model Mohammad Ali Khosravi, Maryam Abbasalipour, Iraj Jafari anarkooli, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5725598/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Spinal cord injury (SCI) can lead to various pathological changes which create an inappropriate environment for repair. The most important of such changes are glial scar and inhibition of neuronal growth in the injured site. Exogenous administration of genes that enhance neuronal survival, synaptic plasticity, and neurotransmission has been considered as a potential approach for treating SCI. Fibromodulin is one of those genes which can decrease TGF-β1 and increase MMP-2 expression, and consequently leads to a reduction in the glial scar, promotes the growth of axons, macrophage activation, and elimination of physical and molecular barriers of neuronal growth that will end with improvement in motor performance. Moreover, bone marrow stromal cells (BMSCs) can be a promising therapeutic strategy for SCI because they can secrete neural growth factors as well as differentiate into neurons. Methods We randomly divided rats into four groups, each consisting of thirteen rats. The first group was administered normal saline, the second group received BMSCs, the third group received BMSCs infected with a beta-galactosidase expressing adenoviral vector, and the fourth group received BMSCs infected with a Fibromodulin expressing adenoviral vector. After inducing spinal cord injury using the weight-dropping method under general anesthesia, BMSCs were injected on the fourth-day post-injury. A Basso, Beattie, and Bresnahan (BBB) score test was conducted for six weeks. At the end of the fourth week, biotin dextran amine (BDA) was intracerebrally injected, and tissue staining was carried out two weeks after the injection. Results The BBB locomotor score test was applied for six weeks. There were significant differences in BBB locomotor scale between the first and the fourthgroups. The mean score of the first group in the sixth week was 5.60, while it was 9.60 for the fourth group. There were significant differences in axon counting between the groups (P<0.000). The average number of axons counted from the first to the fourth group was 87.07, 466.33, 474.13, and 829.40, respectively. Conclusions Consequently, our results highlight the therapeutic potential of the Fibromodulin expressing BMSCs for treating SCI. Fibromodulin Bone marrow stromal cells Gene therapy Cell therapy Spinal cord injury Figures Figure 1 Figure 2 Figure 3 1. Introduction Spinal cord injury is a prevalent disease worldwide. According to a 2013 report by the World Health Organization (WHO), the exact global estimate of spinal cord injury prevalence is unknown, but it occurs approximately in 40 to 80 cases per million populations [ 1 ]. Every year, between 250,000 and 500,000 patients suffer from spinal cord injury globally[ 2 ]. In the United States, the most recent estimate of the annual incidence of spinal cord injury (SCI) is about 54 cases per one million people. This means about 18,000 new SCI cases occur each year, and approximately 282,000 individuals are estimated to be living with SCI [ 3 ]. However, it is estimated to be between 318.45 to 440 per million people in Iran [ 4 ], [ 5 ]. Spinal cord injury can cause paraplegia, which is the loss of muscle function in the lower half of the body, or quadriplegia, which is the partial or total loss of function in all four limbs. This can devastate a patient’s quality of life, life expectancy, and economic burden [ 6 ]. While there have been improvements in quality of life, spinal cord injury remains the leading cause of disability and mortality. Unfortunately, there is currently no fully restorative therapy for SCI, so prevention is key. After spinal cord injury, many pathological events occur, including glial scar formation and inhibition of neuronal growth at the lesion site. Therefore, new therapeutic approaches should be considered that focus on inhibiting glial scar formation and promoting neuronal growth[ 7 ], [ 8 ]. Studies have shown that bone marrow stromal cells (BMSCs) transplantation at the lesion site can play a crucial role in the treatment of spinal cord injury [ 9 ]–[ 13 ]. BMSCs are adult stem cells that originate from bone marrow. One of the clinical benefits of BMSC transplantation therapy is that bone marrow harvesting is more convenient, and there is no immunological issue because the stem cells can be collected from the patients themselves [ 14 ]. BMSC cells also have the potential to differentiate into astrocytes and neurons, and release neurotrophic factors that stimulate axonal growth and promote neuronal survival[ 15 ], [ 16 ]. Therefore, they are considered a promising cellular source for clinical use in autologous grafts for neurological disorders [ 17 ]–[ 20 ]. Fibromodulin (FMOD) is a Keratan sulfate proteoglycan expressed in skin, cornea, and sclera and connective tissues such as cartilage, ligaments, tendons, and dermal tissues. Fibromodulin (FMOD) is a Keratan sulfate proteoglycan expressed in skin, cornea, sclera, cartilage, ligaments, tendons, and dermal tissues. FMOD is a member of the small leucine-rich proteoglycans (SLRPs) family and glycoproteins, including Lumican, Decorin, and Biglycan [ 21 ]–[ 24 ]. FMOD is upregulated in fibrotic, inflammatory, and wound-healing processes in the lung, kidney, liver, and skin, and it is a modulator of TGF-β1 activity [ 22 ], [ 25 ], [ 26 ]. In addition, studies have shown that overexpression of Fibromodulin leads to a reduction in TGF-β1 and an induction in MMP-2 secretion, resulting in a decrease in glial scar formation, an increase in axonal growth, macrophage activation, and the removal of all physical molecular barriers to neural growth. This ultimately leads to an improvement in motor activity [ 27 ]–[ 29 ]. The commonly used viral vectors for gene therapy of SCI are adenovirus, AAV, and lentivirus. AAV and lentivirus lead to the constant expression of the transgene, whereas adenoviral vectors result in transient expression. Since this study is not interested in long-term Fibromodulin expression, adenovirus would be more appropriate for the transient expression of Fibromodulin. Additionally, adenoviral vectors have many advantages, such as high transduction efficiency in quiescent and dividing cell types, high levels of interesting gene expression in the host, and the ability to produce high titers [ 30 ]–[ 36 ]. In this study, we engineered BMSCs to secrete Fibromodulin using an ex vivo adenoviral vector (AdV) transduction technique. These modified BMSCs were transplanted into the injured rat spinal cord, and corticospinal tract (CST) projections were visualized using biotin dextran amine (BDA). Behavioral analysis was performed over a six weeks period using open-field locomotion. 2. Material and methods 2.1 Animals For all experiments, male Sprague-Dawley (SD) rats aged between six to eight weeks and weighing between 210 to 230 grams were used. These rats were obtained from the Razi Vaccine and Serum Research Institute in Iran. Ethical guidelines set by the Ethics Committee of Zanjan University of Medical Sciences were strictly followed in caring for the animals. After spinal cord contusion, all rats received antibiotics treatment (cefazolin 50 mg/kg) and underwent urinary bladder massage two or more times a day until they recovered their ability to urinate spontaneously. 2.2 Isolation of Rat Bone Marrow Stromal Cells (BMSCs) and characterization Bone marrow stromal cells from rats were isolated following a previously described protocol. [ 37 ]. The tibia and femur bones were removed, cleaned, and extracted from rats. The leg's skin, fur, and muscles were peeled off, and the bones were rinsed in 70% ethanol for one minute before being placed in a sterile PBS-filled petri dish. After this stage, all procedures were performed in a biological safety cabinet. The bone ends were cut with scissors, and the bone marrow was flushed into a 50ml tube with sterile PBS using a 22G needle attached to a 5ml syringe. This step was repeated two to three times for each bone. The cell suspension was passed through a 70 µm cell strainer to remove bone debris and blood aggregates. The isolated BMSCs were characterized according to a previously established protocol [ 38 ]. 2.3 Cell culture To prepare the bone marrow cells for culture, 10 ml of fresh DMEM medium (with 10% FBS and 1% pen-strep) was added to the extracted cells. The cells were then seeded in a 25cm2 flask and incubated in a 37℃ and 5% CO2 incubator for 3 hours. After 3 hours, the medium was replaced with a fresh DMEM medium. As the BMSCs can adhere to the surface of the culture flask, the suspension cells were excluded from this step. The culture medium was replaced with fresh medium every day for the first three days and then every 3 or 4 days. After approximately 2–3 weeks, the cells had reached efficient confluences and could be subcultured when they reached a confluence of ≥ 60%. The culture medium was removed, and the monolayer cells were rinsed with PBS. 2ml Trypsin-EDTA was added to the washed cells and incubated for 2–5 minutes at 37℃ until the cells were detached. To inhibit trypsin action, 5–10 ml media containing serum were added. The cells were collected in a tube and centrifuged for approximately 5 minutes at 400g. Then, the cell pellet was resuspended in fresh medium and dispensed into two flasks. 2.4 Adenoviral vector For this study, replication-defective adenoviruses containing bovine Fibromodulin (Ad5-FMOD), and adenoviruses containing the β-galactosidase gene (Ad5-LacZ) were kindly gifted by Dr. Paul Kingstone (The University of Manchester, U.K) [ 39 ]. 2.5 BMSCs transduction by Ad5-FMOD vector To determine the Adenoviral titration, a plaque assay was conducted in HEK 293 cells. Two days before transduction, BMSC cells were plated in a 24-well plate with 2 × 105 cells per well. The Ad5-FMOD and Ad5-LacZ were diluted in RPMI 1640 serum-free culture media (Gibco, Invitrogen) and added to the cells with a multiplicity of infection (MOI) of 100 pfu/cell. The cells were then incubated at 37°C. Four hours after transduction, the media was removed, and the cells were washed with PBS and cultured again in fresh medium with 15% FBS for 48h. 2.6 Evaluation of Fibromodulin expression by RT-PCR The Qiagen RNeasy Mini Kit (Cat.No: 74104) was used for extracting total RNA from transduced cells, following the manufacturer's instructions. A total of 1 µg of RNA was utilized to generate cDNA, using the Qiagen One-Step RT-PCR cDNA Synthesis Kit as per the manufacturer's specifications. The RT-PCR was carried out with the following primers: FMOD forward primer (FMF1) 5′-TGAAGGCAGCACCTGACCGC-3′, FMOD reverse primer (FMR1) 5′-ACGCCTTGGCTTCTCCTGCC-3′ (189bp). β-Actin forward primer 5′ AAGCAGGAGTATGACGAGTC-3′, β-Actin reverse primer 5′ CCGTTCCAGTTTTTAAATCC-3′ (207bp). The PCR was performed for 40 cycles under the following conditions: 30 min at 50°C, 15 min at 95°C, 45 sec at 94°C, 45 sec at 63°C, 1 min at 72°C. 2.7 Spinal Cord Injury Rat Model To perform spinal cord injury, a "weight dropping" method was utilized. Thirteen rats were randomly assigned to each of the four groups. All rats were anesthetized by intraperitoneal injection of 87 mg of ketamine per kg of body weight and 10 mg of xylazine per kg of body weight. To prevent dryness of the cornea, a drop of mineral oil was used. The thoracic area was shaved and disinfected with povidone-iodine solution. Then, under a microscope, laminectomy surgery was performed on the tenth thoracic vertebra (T10). The spinal cord was injured by dropping a 10-gram metal rod from a 50-mm distance onto the exposed spinal cord (T10 vertebra). The wound was closed using chromic catgut (4/0) for the muscle and nylon suture (3/0) for the skin. After the surgery, rats were given Cefazolin (50 mg/kg BW/day intramuscular) for three days and placed on a 37°C heating blanket overnight [ 40 ]–[ 42 ]. 2.8 Bone Marrow Stromal Cells Transplantation (BMSCT) Four days following a spinal cord contusion, 2x105 transduced cells were suspended in phosphate-buffered saline (PBS). Rats were then fixed in a stereotaxic device, and the cells were injected using a 5µl Hamilton syringe to a depth of 1.5mm in the caudal border of the lesion site for 120 seconds. The first group of rats received normal saline, the second group was administered with BMSCs, the third group was given BMSCs infected with adenovirus expressing beta-galactosidase, and the fourth group received BMSCs infected with adenovirus expressing Fibromodulin. 2.9 Behavioral analysis The behavioral functions of the animals were evaluated one week after cell injection. The locomotor BBB test was used to assess their progress for six weeks after surgery. The BBB test is an open field score that ranges from complete hind limb paralysis (Zero) to normal movement (Twenty-one). Two observers, who were unaware of the treatment, scored the animals according to the BBB scale [ 43 ], [ 44 ]. 2.10 BDA anterograde CST tracing To trace the corticospinal tract (CST), anterograde tracing was performed using BDA, which was administered as per standard procedures. The experiment involved 12 rats, with three rats randomly selected from each group. The rats were anesthetized, and their skulls were secured using a stereotaxic device. The surgical area was cleansed and sanitized with an iodine swab. A craniotomy was then performed, creating a hole approximately 1.0 mm in diameter and depth, at a location 2 mm lateral and 1.6 mm caudal to the bregma. Using a 5µl Hamilton syringe, a slow injection of 1 µl of 10% BDA (Life Technologies, Cat N: D-1956) was administered into the cerebral motor cortex at a depth of 1.5 mm for 160 seconds. The BDA was injected two weeks before the rats were sacrificed [ 45 ]. 2.11 Histological procedures To prepare for analysis, one centimeter of the spinal cord with the lesion at the midpoint was cut and embedded in paraffin. The embedded spinal cords were cut into serial transverse sections, each 5-µm thick with a 200 µm interval, using a freezing microtome (Rotary Microtome, YD-2508). BDA labeling was performed as previously described. Briefly, the sections were rinsed in 0.1M Tris-buffered saline (TBS; pH 7.4) or PBS and treated with 0.6% hydrogen peroxide in TBS or PBS for 30 minutes to inhibit endogenous peroxidase activity. They were then incubated with avidin-biotin-peroxidase complex (VECTASTAIN® Elite® ABC HRP Kit; PK-6100, USA). After washing the sections, they were treated in diaminobenzidine tetrahydrochloride (DAB) and nickel chloride until the production of a dark reaction. Sections were photographed under a Nikon microscope (×40). The extent of the DAB labeled fibers in each section was quantified in a blinded manner using Scion Image software. 2.12 Statistical analysis Data were analyzed using SPSS v16 (Chicago, Inc., USA) and expressed as mean ± S.E.M. Statistical analysis involved one-way and two-way ANOVA, followed by post-hoc analysis with the Tukey test. 3. Results 3.1 Transferred gene expression in vitro To assess the expression level of Fibromodulin mRNA in vitro, reverse transcription-PCR (RT-PCR) was performed. The results showed that Fibromodulin mRNA expression was confirmed in the fourth group, as compared to the control groups (as shown in Fig. 1 ). The biological activity of an Adenoviral vector carrying the Fibromodulin gene was confirmed via a bioassay of TGF-β activity, as previously reported by P Ranjzad et al. 3.2 Recovery of hind limb function The behavioral analysis started one week after injecting cells and continued weekly for 6 weeks after spinal cord injury (see Fig. 2 ). To determine the locomotor recovery of rats, the BBB locomotion score was used, which considers the early (BBB score from 0 to 7), intermediate (8–13) and late phases (14–21) of recovery [ 44 ]. According to statistical analysis, there were significant differences between groups during the 3rd, 5th, and 6th weeks. The probability values (p-values) were p = 0.002, p = 0.047, and p = 0.006, respectively. However, no differences in BBB scores were found between all groups at other time points (p < 0.05). No significant differences were observed until 1 week after injury between groups (p-value = 0.325). In the third week, the first signs of locomotor function recovery were observed. There were significant differences between the fourth and control groups (p-value = 0.00). At week six, the average score for the control group (group 1) was 5.60 ± 1.140, for the second group was 8.00, for the third group was 8.50 ± 0.707, and for the fourth group was 9.60 ± 2.675. The average BBB score for the fourth group was significantly higher than the control group (p-value = 0.03) over six weeks, indicating that the Fibromodulin gene had a significant effect on functional recovery. 3.3 Quantification of CST axons Data collected from BDA anterograde tracing of CST fibers were analyzed to examine the diffusion pattern of sprouting CST fibers after spinal cord injury. The lowest axon count was obtained in the control group, with an average of 87.07 ± 46.75. The average number of axons was 466.33 ± 146.959 in group 2, 474.13 ± 109.149 in group 3, and 829.40 ± 139.006 in group 4. Groups 2, 3, and 4 showed significantly higher numbers of axons compared to the control group (P ≤ 0.001). The number of axons in group 4 was statistically significant versus groups 2 and 3 (P ≤ 0.001) (Fig. 3 ). 4. Discussion A spinal cord injury (SCI) is a lesion on any part of the spinal cord that leads to short-term or steady-state changes in its normal motor, sensory, or autonomic function. Unfortunately, damaged axons do not generally regenerate and so far, there is no efficient clinically approved strategy to cure SCI [ 6 ], [ 46 ]. FMOD, a member of the SLRP family, is recognized for its interaction with collagen fibrils and the configuration of the extracellular matrix. Previous studies have reported that FMOD plays a significant role in cell fate determination, fetal-type scarless wound healing stimulates adult wound closure, and decreases scar formation [ 25 ], [ 47 ]–[ 49 ]. Recent years have seen stem cell transplantation, such as bone marrow mesenchymal cells (BMSCs), embryonic stem cells (ESCs), and umbilical cord blood stem cells, being used to treat spinal cord injury (SCI). This new strategy has shown promise in activating neuroregeneration and restoring spinal cord functions [ 50 ], [ 51 ]. BMSCs, in particular, have demonstrated good differentiation potential and neural recovery. They are capable of differentiating into glial cells and neurons, repairing the myelin sheath of injured axons, and regenerating nerve fibers [ 20 ], [ 52 ]. Additionally, BMSCs produce various trophic factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and cytokines such as IL-6 and stem cell factor (SCF), and IGF-1, which are effective in promoting neural protection or regeneration [ 53 ], [ 54 ]. Transplanted BMSCs produce neurotrophic factors such as BDNF and GDNF that can reduce neuronal cell death protect injured neural tissue and promote axon regrowth, respectively [ 54 ]. These factors make BMSC transplantation a promising method for treating SCI. In 2000, Chopp and colleagues demonstrated motor improvement using BMSCs transplantation at the site of the injury in rat models [ 55 ]. In 2003, Wu and colleagues investigated bone marrow stromal cell grafting in the lesion site. Transplanted BMSCs stimulated the regeneration of the injured spinal cord by raising tissue repair of the lesion and resulted in smaller cavities than in controls. In another study, Ide and colleagues reduced cavity formation and myelinated injured axons, and increased the BBB score to 9.8 (compared to 5.5–5.7 in the control group) using direct transplantation of BMSCs in the lesion site two weeks post-injury in subacute-spinal-cord injury [ 56 ]. To investigate the therapeutic effects of Fibromodulin in combination with BMSCs in a rat spinal cord injury model, the present study transduced BMSCs with adenoviral vectors carrying the Fibromodulin gene. These modified cells were then transplanted into SCI, leading to promoting axonal regeneration and functional recovery. The three BMSCs transplanted groups (BMSCs, BMSCs -LacZ, and BMSCs -Fibromodulin) showed significant axonal regeneration when compared with rats in the control group. This is consistent with previous reports showing that BMSCs injection increases the capability of axon regrowth at the injury site [ 52 ], [ 55 ], [ 57 ]–[ 60 ]. The study found that transplantation of Fibromodulin-expressing BMSCs into the spinal cord four days after injury significantly improved functional outcomes, as evaluated on the BBB test. Significant recovery of functional outcomes extended up to 6 weeks after transplantation. BBB locomotor scaling score results indicated significant scores in weeks 2, 3, 4, 5, and 6 after injection. Behavioral follow-up was performed 6 weeks post-injury. There was no statistical difference until 1 week after injury between groups (p value = 0.325). The average score in the 6th week was 5.60 ± 1.140, 8.00, 8.50 ± 0.707, and 9.60 ± 2.675 for groups 1st, 2nd, 3rd, and 4th, respectively. The fourth group had a statistically significant BBB score in comparison with the control group (p value = 0.03) in the sixth week, indicating that the Fibromodulin gene had a significant effect on functional recovery. In the third week, the first signs of locomotor function recovery were observed, and they were statistically significant between the fourth and control groups (p value = 0.00). 5. Conclusions Our study suggests that Fibromodulin may have a significant role in promoting axonal growth after severe injury. As there is no similar study to compare the results with, our focus was to investigate the potential of Fibromodulin for gene therapy of spinal cord injury (SCI) for the first time. Our study also confirmed the positive effect of bone marrow cell therapy combined with gene therapy. Therefore, our findings indicate that the combination of cell therapy (using BMSCs) and gene therapy (using Fibromodulin) can be considered a promising approach for gene therapy of SCI. Declarations Animal Ethics "All experimental protocols were undertaken in compliance with the Institutional Animal Care & Use Committee (IACUC) standards and approved by the Zanjan University of Medical Sciences Ethics Review Board". Ethics approval and consent to participate Ethical guidelines set by the Ethics Committee of Zanjan University of Medical Sciences were strictly followed in caring for the animals. Consent for publication Not applicable. This manuscript does not include any individual person’s data in any form (including individual details, images, or videos). Availability of data and material Data will be made available upon reasonable request. Competing interests The authors declare that they have no conflicts of interest. Funding This work was supported by the Zanjan University of Medical Sciences [grant number 88112003]; and council for Stem cell Science and Technology [grant number 700/391] (Presidency of the Islamic Republic of Iran Vice-presidency for Science and Technology). Authors' contributions Conception and design: MAK, AB, PR, PAK. Acquisition of data: MAK, MA, IJA. Analysis and interpretation of data: SM, MAK, MA, And AB. Drafting the article: MAK, MA. Critically revising the article: IJA, SM, PR, and PAK. Reviewed submitted version of manuscript: MAK, MA, IJA, SM, PR, PAK, and AB. Acknowledgements This work was supported by Zanjan University of Medical Sciences, we also wish to thank Prof. Paul Kingston and Parisa Ranjzad, Vascular Gene Therapy Unit, Research School of Clinical & Laboratory Sciences, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK, for giving us adenoviral vectors. References M. G. F. Christopher D. Witiw, “Acute Spinal Cord Injury,” J. Spinal Disord. Tech. , vol. 28, no. 6, pp. 202–210, Jul. 2015, doi: 10.1097/bsd.0000000000000287. J. Bennett, J. M. Das, and P. D. Emmady, “Spinal Cord Injuries,” StatPearls , May 2022. National Spinal Cord Injury Statistical Center, “Spinal cord injury facts and figures at a glance.,” J. Spinal Cord Med. , vol. 36, no. 1, pp. 1–2, 2013, doi: 10.1179/1079026813Z.000000000136. A. Singh, L. Tetreault, S. Kalsi-Ryan, A. Nouri, and M. G. Fehlings, “Global prevalence and incidence of traumatic spinal cord injury.,” Clin. Epidemiol. , vol. 6, pp. 309–31, 2014, doi: 10.2147/CLEP.S68889. S. B. Jazayeri et al. , “Prevalence of Spinal Cord Injury in Iran: A 3-Source Capture-Recapture Study,” Neuroepidemiology , vol. 45, no. 1, pp. 28–33, Aug. 2015, doi: 10.1159/000435785. S. Thuret, L. D. F Moon, and F. H. Gage, “Autonomic dysreflexia Therapeutic interventions after spinal cord injury,” 2006, doi: 10.1038/nrn1955. R. Lima, A. Monteiro, A. J. Salgado, S. Monteiro, and N. A. Silva, “Pathophysiology and Therapeutic Approaches for Spinal Cord Injury,” Int. J. Mol. Sci. 2022, Vol. 23, Page 13833 , vol. 23, no. 22, p. 13833, Nov. 2022, doi: 10.3390/IJMS232213833. A. Anjum et al. , “Spinal Cord Injury: Pathophysiology, Multimolecular Interactions, and Underlying Recovery Mechanisms,” Int. J. Mol. Sci. , vol. 21, no. 20, pp. 1–35, Oct. 2020, doi: 10.3390/IJMS21207533. R. Stewart and S. Przyborski, “Non-neural adult stem cells: tools for brain repair?,” BioEssays , vol. 24, no. 8, pp. 708–713, Aug. 2002, doi: 10.1002/bies.10124. C. S. Padovan et al. , “Expression of neuronal markers in differentiated marrow stromal cells and CD133+ stem-like cells.,” Cell Transplant. , vol. 12, no. 8, pp. 839–48, 2003. C. Lv, T. Zhang, K. Li, and K. Gao, “Bone marrow mesenchymal stem cells improve spinal function of spinal cord injury in rats via TGF-β/Smads signaling pathway,” Exp. Ther. Med. , 2020, doi: 10.3892/etm.2020.8640. S. Bai, H. Zhou, and L. Wu, “Bone marrow stromal cells improved functional recovery in spinal cord injury rats partly via the Toll-like receptor-4/nuclear factor-κB signaling pathway,” Exp. Ther. Med. , vol. 17, no. 1, p. 444, Oct. 2019, doi: 10.3892/ETM.2018.6907. O. Honmou et al. , “Intravenous infusion of auto serum-expanded autologous mesenchymal stem cells in spinal cord injury patients: 13 case series,” Clin. Neurol. Neurosurg. , vol. 203, p. 106565, Apr. 2021, doi: 10.1016/J.CLINEURO.2021.106565. J.-B. Lee et al. , “A pre-clinical assessment model of rat autogeneic bone marrow stromal cell transplantation into the central nervous system.,” Brain Res. Brain Res. Protoc. , vol. 14, no. 1, pp. 37–44, Nov. 2004, doi: 10.1016/j.brainresprot.2004.09.004. G. Liu, Y. Wu, F. Kong, S. Ma, L. Fu, and J. Geng, “BMSCs differentiated into neurons, astrocytes and oligodendrocytes alleviated the inflammation and demyelination of EAE mice models,” bioRxiv , p. 2020.11.16.384354, Nov. 2020, doi: 10.1101/2020.11.16.384354. H. Cheng et al. , “Cyclic Strain and Electrical Co-stimulation Improve Neural Differentiation of Marrow-Derived Mesenchymal Stem Cells,” Front. Cell Dev. Biol. , vol. 9, p. 624755, May 2021, doi: 10.3389/FCELL.2021.624755/BIBTEX. A. M. Parr, C. H. Tator, and A. Keating, “Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury,” Bone Marrow Transplant. , vol. 40, pp. 609–619, 2007, doi: 10.1038/sj.bmt.1705757. P. Bossolasco et al. , “Neuro-glial differentiation of human bone marrow stem cells in vitro,” 2005, doi: 10.1016/j.expneurol.2004.12.013. R. Io Garc Ia, J. Aguiar, E. Alberti, K. De La Cu Etara, and N. Pav, “Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors,” doi: 10.1016/j.bbrc.2004.02.111. T. R. Brazelton, F. M. Rossi, G. I. Keshet, and H. M. Blau, “From marrow to brain: expression of neuronal phenotypes in adult mice.,” Science , vol. 290, no. 5497, pp. 1775–9, Dec. 2000. S. Chakravarti, J. Paul, L. Roberts, I. Chervoneva, A. Oldberg, and D. E. Birk, “Ocular and scleral alterations in gene-targeted lumican-fibromodulin double-null mice,” Investig. Ophthalmol. Vis. Sci. , vol. 44, no. 6, pp. 2422–2432, 2003, doi: 10.1167/iovs.02-0783. K. Andenæs et al. , “The extracellular matrix proteoglycan fibromodulin is upregulated in clinical and experimental heart failure and affects cardiac remodeling,” PLoS One , vol. 13, no. 7, pp. 1–20, 2018, doi: 10.1371/journal.pone.0201422. F. Zhao, Y. Bai, X. Xiang, and X. Pang, “The role of fibromodulin in inflammatory responses and diseases associated with inflammation,” Front. Immunol. , vol. 14, p. 1191787, Jul. 2023, doi: 10.3389/FIMMU.2023.1191787/BIBTEX. Z. Zheng, H. S. Granado, and C. Li, “Fibromodulin, a Multifunctional Matricellular Modulator,” https://doi.org/10.1177/00220345221138525 , vol. 102, no. 2, pp. 125–134, Dec. 2022, doi: 10.1177/00220345221138525. Z. Zheng et al. , “Fibromodulin reduces scar formation in adult cutaneous wounds by eliciting a fetal-like phenotype,” Signal Transduct. Target. Ther. , vol. 2, p. 17050, Oct. 2017, doi: 10.1038/sigtrans.2017.50. Z. Zheng et al. , “Fibromodulin-Deficiency Alters Temporospatial Expression Patterns of Transforming Growth Factor-β Ligands and Receptors during Adult Mouse Skin Wound Healing,” PLoS One , vol. 9, no. 3, p. e90817, Mar. 2014, doi: 10.1371/journal.pone.0090817. A. Stoff et al. , “Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds,” J. Mol. Med. , vol. 85, no. 5, pp. 481–496, May 2007, doi: 10.1007/s00109-006-0148-z. M. Kohta, E. Kohmura, and T. Yamashita, “Inhibition of TGF-b1 promotes functional recovery after spinal cord injury,” doi: 10.1016/j.neures.2009.08.017. J. C. Hsu et al. , “Matrix metalloproteinase-2 facilitates wound healing events that promote functional recovery after spinal cord injury.,” undefined , 2006. M. Koda et al. , “Adenovirus Vector-Mediated In Vivo Gene Transfer of Brain-Derived Neurotrophic Factor (BDNF) Promotes Rubrospinal Axonal Regeneration and Functional Recovery after Complete Transection of the Adult Rat Spinal Cord,” J. Neurotrauma , vol. 21, no. 3, pp. 329–337, Mar. 2004, doi: 10.1089/089771504322972112. Y. Zhang et al. , “Enhanced Adenoviral Gene Delivery to Motor and Dorsal Root Ganglion Neurons Following Injection Into Demyelinated Peripheral Nerves,” doi: 10.1002/jnr.22394. Y. Liu et al. , “Application of recombinant adenovirus for in vivo gene delivery to spinal cord,” Brain Res. , vol. 768, no. 1–2, pp. 19–29, Sep. 1997, doi: 10.1016/S0006-8993(97)00587-8. A. P. Tosolini and R. Morris, “Viral-mediated gene therapy for spinal cord injury (SCI) from a translational neuroanatomical perspective.,” Neural Regen. Res. , vol. 11, no. 5, pp. 743–4, May 2016, doi: 10.4103/1673-5374.182698. A. A. Abdellatif et al. , “Gene delivery to the spinal cord: comparison between lentiviral, adenoviral, and retroviral vector delivery systems.,” J. Neurosci. Res. , vol. 84, no. 3, pp. 553–67, Aug. 2006, doi: 10.1002/jnr.20968. A. O. Sosnovtseva et al. , “Recombinant Adenoviruses for Delivery of Therapeutics Following Spinal Cord Injury,” Front. Pharmacol. , vol. 12, Jan. 2021, doi: 10.3389/FPHAR.2021.777628. F. C. Zhu, D. M. Jiang, M. H. Zhang, B. Zhao, C. He, and J. Yang, “Adenovirus vector-mediated in vivo gene transfer of nuclear factor erythroid-2p45-related factor 2 promotes functional recovery following spinal cord contusion,” Mol. Med. Rep. , vol. 20, no. 5, pp. 4285–4292, 2019, doi: 10.3892/mmr.2019.10687. M. Soleimani and S. Nadri, “A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow,” Nat. Protoc. , vol. 4, no. 1, pp. 102–106, Jan. 2009, doi: 10.1038/nprot.2008.221. M. Shahrezaie et al. , “Improved stem cell therapy of spinal cord injury using GDNF-overexpressed bone marrow stem cells in a rat model,” Biologicals , vol. 50, pp. 73–80, Nov. 2017, doi: 10.1016/j.biologicals.2017.08.009. P. Ranjzad, H. K. Salem, and P. A. Kingston, “Adenovirus-mediated gene transfer of fibromodulin inhibits neointimal hyperplasia in an organ culture model of human saphenous vein graft disease,” Gene Ther. , vol. 16, no. 9, pp. 1154–1162, Sep. 2009, doi: 10.1038/gt.2009.63. B. T. Kalish and M. J. Whalen, “Weight Drop Models in Traumatic Brain Injury,” Humana Press, New York, NY, 2016, pp. 193–209. J. A. GRUNER, “A Monitored Contusion Model of Spinal Cord Injury in the Rat,” J. Neurotrauma , vol. 9, no. 2, pp. 123–128, Jan. 1992, doi: 10.1089/neu.1992.9.123. Y. Bomstein et al. , “Features of skin-coincubated macrophages that promote recovery from spinal cord injury,” J. Neuroimmunol. , vol. 142, no. 1–2, pp. 10–16, Sep. 2003, doi: 10.1016/S0165-5728(03)00260-1. T. E. P. de Barros Filho and A. E. I. S. Molina, “Analysis of the sensitivity and reproducibility of the Basso, Beattie, Bresnahan (BBB) scale in Wistar rats.,” Clinics (Sao Paulo). , vol. 63, no. 1, pp. 103–8, Feb. 2008. D. M. BASSO, M. S. BEATTIE, and J. C. BRESNAHAN, “A Sensitive and Reliable Locomotor Rating Scale for Open Field Testing in Rats,” J. Neurotrauma , vol. 12, no. 1, pp. 1–21, Feb. 1995, doi: 10.1089/neu.1995.12.1. I. A. Ferguson, C. Xian, E. Barati, and R. A. Rush, “Comparison of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury.,” J. Neurosci. Methods , vol. 109, no. 2, pp. 81–9, Aug. 2001. S. M. Willerth and S. E. Sakiyama-Elbert, “Cell therapy for spinal cord regeneration.,” Adv. Drug Deliv. Rev. , vol. 60, no. 2, pp. 263–76, Jan. 2008, doi: 10.1016/j.addr.2007.08.028. Z. Zheng et al. , “Fibromodulin Is Essential for Fetal-Type Scarless Cutaneous Wound Healing.,” Am. J. Pathol. , vol. 186, no. 11, pp. 2824–2832, 2016, doi: 10.1016/j.ajpath.2016.07.023. J. Jian et al. , “Fibromodulin promoted in vitro and in vivo angiogenesis,” Biochem. Biophys. Res. Commun. , vol. 436, no. 3, pp. 530–535, Jul. 2013, doi: 10.1016/J.BBRC.2013.06.005. Z. Zheng et al. , “Fibromodulin Enhances Angiogenesis during Cutaneous Wound Healing,” Plast. Reconstr. Surg. Glob. Open , vol. 2, no. 12, p. e275, Dec. 2014, doi: 10.1097/GOX.0000000000000243. R. Vawda, J. Wilcox, and M. Fehlings, “Current stem cell treatments for spinal cord injury.,” Indian J. Orthop. , vol. 46, no. 1, pp. 10–8, Jan. 2012, doi: 10.4103/0019-5413.91629. A. J. Mothe and C. H. Tator, “Advances in stem cell therapy for spinal cord injury.,” J. Clin. Invest. , vol. 122, no. 11, pp. 3824–34, Nov. 2012, doi: 10.1172/JCI64124. M. Sasaki, O. Honmou, Y. Akiyama, T. Uede, K. Hashi, and J. D. Kocsis, “Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons,” Glia , vol. 35, no. 1, pp. 26–34, 2001, doi: 10.1002/glia.1067. C. Ide et al. , “Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat,” Brain Res. , vol. 1332, pp. 32–47, May 2010, doi: 10.1016/J.BRAINRES.2010.03.043. M. Enomoto, “The future of bone marrow stromal cell transplantation for the treatment of spinal cord injury,” Neural Regen. Res. , vol. 10, no. 3, p. 383, Mar. 2015, doi: 10.4103/1673-5374.153684. M. Chopp et al. , “Spinal cord injury in rat: Treatment with bone marrow stromal cell transplantation,” Neuroreport , vol. 11, no. 13, pp. 3001–3005, 2000, doi: 10.1097/00001756-200009110-00035. S. Wu et al. , “Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord,” J. Neurosci. Res. , vol. 72, no. 3, pp. 343–351, May 2003, doi: 10.1002/jnr.10587. J. W. Kim, K. Y. Ha, J. N. Molon, and Y. H. Kim, “Bone marrow-derived mesenchymal stem cell transplantation for chronic spinal cord injury in rats: Comparative study between intralesional and intravenous transplantation,” Spine (Phila. Pa. 1976). , vol. 38, no. 17, pp. 1065–1074, 2013, doi: 10.1097/BRS.0b013e31829839fa. C. Ide et al. , “Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat,” Brain Res. , vol. 1332, pp. 32–47, May 2010, doi: 10.1016/j.brainres.2010.03.043. N. Nakano et al. , “Effects of Bone Marrow Stromal Cell Transplantation through CSF on the Subacute and Chronic Spinal Cord Injury in Rats,” PLoS One , vol. 8, no. 9, p. e73494, Sep. 2013, doi: 10.1371/journal.pone.0073494. L. Lin, H. Lin, S. Bai, L. Zheng, and X. Zhang, “Bone marrow mesenchymal stem cells (BMSCs) improved functional recovery of spinal cord injury partly by promoting axonal regeneration,” Neurochem. Int. , vol. 115, pp. 80–84, May 2018, doi: 10.1016/J.NEUINT.2018.02.007. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5725598","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":415113608,"identity":"cf2545af-45aa-4631-8472-8649995fb87e","order_by":0,"name":"Mohammad Ali Khosravi","email":"","orcid":"","institution":"Zanjan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Ali","lastName":"Khosravi","suffix":""},{"id":415113609,"identity":"7e0996de-db9f-45ac-b06c-802818cc91ee","order_by":1,"name":"Maryam Abbasalipour","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Abbasalipour","suffix":""},{"id":415113610,"identity":"eabafe7e-ac01-41dc-828f-49e2f1ba854c","order_by":2,"name":"Iraj Jafari anarkooli","email":"","orcid":"","institution":"Zanjan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Iraj","middleName":"Jafari","lastName":"anarkooli","suffix":""},{"id":415113611,"identity":"f8372d1b-1dc7-4fe6-a5d0-c58e79862bf6","order_by":3,"name":"Saeideh Mazloomzadeh","email":"","orcid":"","institution":"Zanjan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Saeideh","middleName":"","lastName":"Mazloomzadeh","suffix":""},{"id":415113612,"identity":"c694923d-74d8-4fdb-a026-727c3dce9ff8","order_by":4,"name":"Parisa Ranjzad","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Parisa","middleName":"","lastName":"Ranjzad","suffix":""},{"id":415113613,"identity":"b98a90f7-e96a-45b3-93b5-4744e4d36f58","order_by":5,"name":"Paul A Kingston","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"A","lastName":"Kingston","suffix":""},{"id":415113614,"identity":"ede04668-ab0a-44fe-9cf5-6e2fbcf69fb7","order_by":6,"name":"Alireza Biglari","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie3PMWrDMBSA4SceyMujXm2SOFeQ8Z6eJWT1YOgUmrgpAvcKgdxDc8BQLyZzIEvdnMCUQAMdItlktToWqh/xwEIfsgBcrr+YxzYIAsYA7PVDf9ODlWBPSBMpDOF20i1DgAdmw0p8icVXlq3J35XF8pLOxhyw+TwOkKBkcrcVFQWHeXGaqIX+MZ4k6dA1miCJd4KaFadQoSbER0NkeidTTZ5C9WInoicrEpqwVpV2EhuyFXuKayZHTFXE0fKWqHo7Y/aTR1HtNe1VPT/6nmzOg8/vK7uJ1E37cVPeTfb9u9Mul8v1z7oB+84/kn7v3LIAAAAASUVORK5CYII=","orcid":"","institution":"Zanjan University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Biglari","suffix":""}],"badges":[],"createdAt":"2024-12-28 10:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5725598/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5725598/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80688292,"identity":"9d559232-07df-48e8-a6e1-cd192438158f","added_by":"auto","created_at":"2025-04-16 04:28:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":128049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the expression of Fibromodulin in BMSCs.\u003c/strong\u003e RT-PCR of Fibromodulin mRNA of cells infected by Ad5-FMOD. There is a band of 189 bp on the lane of BMSCs+FMOD, and a band of 207bp for β-actin. Marker: 100 bp.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5725598/v1/86544f44d63cac236c2d6699.png"},{"id":80688616,"identity":"2a7e10aa-b7dc-4f29-87c7-8ed08846df9c","added_by":"auto","created_at":"2025-04-16 04:36:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional recovery.\u003c/strong\u003e Open field locomotor evaluation using BBB scale was recorded every week after transplantation. Statistical analysis indicates that BBB scales in the BMSCs+fibromodulin transplantation group are significantly higher than those in the control group (P \u0026lt; 0.05).Values represent mean ± S.E.M. * P≤0.05 compared with the control group. S.E.M. standard error of the mean.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5725598/v1/2e8df88a4b02ce74c19cd55c.png"},{"id":80688295,"identity":"cb6a6139-4eee-48da-b217-314d15f0ae31","added_by":"auto","created_at":"2025-04-16 04:28:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":400413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative analysis of CST axons at 6 weeks after transplantation.\u003c/strong\u003e Effect of BMSCs on axonal regeneration at 6 weeks after spinal cord injury (SCI). (A–D) Measurements of BDA -labeled axons (brown spots) at the lesion site, as were obtained from transversal spinal cord sections from all experimental groups and were presented as numbers of axons (mean ±S.D.). (A) Rats treated with PBS as a control group, (B) rats treated with BMSCs, (C) rats treated with BMSCs infected with adenovirus expressing beta-galactosidase, and (D) rats treated with BMSCs infected with adenovirus expressing Fibromodulin. (E) Quantitative data showing the numbers of BDA-labeled axons per section (n = 12/group). Data were shown as mean ± S.E.M. ***P \u0026lt; 0.001, as compared with the control group. Significant differences were detected in the BMSCs-fibromodulin implanted group compared with the control group (***P\u0026lt;0.001), BMSCs-LacZ implanted group and BMSCs implanted group (*P\u0026lt;0.05). Moreover, there are significant differences between BMSCs-fibromodulin group and BMSCs-LacZ and BMSCs groups (*P\u0026lt;0.05). However, there is no statistical difference between BMSCs-LacZ and BMSCs groups (P value 0.8925).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5725598/v1/d8c15f24f3e2830ccec93f39.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gene therapy of spinal cord injury using gene-modified Bone Marrow Stromal Cells with Fibromodulin expressing adenoviral vector in a rat SCI model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpinal cord injury is a prevalent disease worldwide. According to a 2013 report by the World Health Organization (WHO), the exact global estimate of spinal cord injury prevalence is unknown, but it occurs approximately in 40 to 80 cases per million populations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Every year, between 250,000 and 500,000 patients suffer from spinal cord injury globally[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In the United States, the most recent estimate of the annual incidence of spinal cord injury (SCI) is about 54 cases per one million people. This means about 18,000 new SCI cases occur each year, and approximately 282,000 individuals are estimated to be living with SCI [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, it is estimated to be between 318.45 to 440 per million people in Iran [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Spinal cord injury can cause paraplegia, which is the loss of muscle function in the lower half of the body, or quadriplegia, which is the partial or total loss of function in all four limbs. This can devastate a patient\u0026rsquo;s quality of life, life expectancy, and economic burden [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While there have been improvements in quality of life, spinal cord injury remains the leading cause of disability and mortality. Unfortunately, there is currently no fully restorative therapy for SCI, so prevention is key. After spinal cord injury, many pathological events occur, including glial scar formation and inhibition of neuronal growth at the lesion site. Therefore, new therapeutic approaches should be considered that focus on inhibiting glial scar formation and promoting neuronal growth[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Studies have shown that bone marrow stromal cells (BMSCs) transplantation at the lesion site can play a crucial role in the treatment of spinal cord injury [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. BMSCs are adult stem cells that originate from bone marrow. One of the clinical benefits of BMSC transplantation therapy is that bone marrow harvesting is more convenient, and there is no immunological issue because the stem cells can be collected from the patients themselves [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. BMSC cells also have the potential to differentiate into astrocytes and neurons, and release neurotrophic factors that stimulate axonal growth and promote neuronal survival[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, they are considered a promising cellular source for clinical use in autologous grafts for neurological disorders [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Fibromodulin (FMOD) is a Keratan sulfate proteoglycan expressed in skin, cornea, and sclera and connective tissues such as cartilage, ligaments, tendons, and dermal tissues. Fibromodulin (FMOD) is a Keratan sulfate proteoglycan expressed in skin, cornea, sclera, cartilage, ligaments, tendons, and dermal tissues. FMOD is a member of the small leucine-rich proteoglycans (SLRPs) family and glycoproteins, including Lumican, Decorin, and Biglycan [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. FMOD is upregulated in fibrotic, inflammatory, and wound-healing processes in the lung, kidney, liver, and skin, and it is a modulator of TGF-β1 activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, studies have shown that overexpression of Fibromodulin leads to a reduction in TGF-β1 and an induction in MMP-2 secretion, resulting in a decrease in glial scar formation, an increase in axonal growth, macrophage activation, and the removal of all physical molecular barriers to neural growth. This ultimately leads to an improvement in motor activity [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The commonly used viral vectors for gene therapy of SCI are adenovirus, AAV, and lentivirus. AAV and lentivirus lead to the constant expression of the transgene, whereas adenoviral vectors result in transient expression. Since this study is not interested in long-term Fibromodulin expression, adenovirus would be more appropriate for the transient expression of Fibromodulin. Additionally, adenoviral vectors have many advantages, such as high transduction efficiency in quiescent and dividing cell types, high levels of interesting gene expression in the host, and the ability to produce high titers [\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, we engineered BMSCs to secrete Fibromodulin using an ex vivo adenoviral vector (AdV) transduction technique. These modified BMSCs were transplanted into the injured rat spinal cord, and corticospinal tract (CST) projections were visualized using biotin dextran amine (BDA). Behavioral analysis was performed over a six weeks period using open-field locomotion.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eFor all experiments, male Sprague-Dawley (SD) rats aged between six to eight weeks and weighing between 210 to 230 grams were used. These rats were obtained from the Razi Vaccine and Serum Research Institute in Iran. Ethical guidelines set by the Ethics Committee of Zanjan University of Medical Sciences were strictly followed in caring for the animals. After spinal cord contusion, all rats received antibiotics treatment (cefazolin 50 mg/kg) and underwent urinary bladder massage two or more times a day until they recovered their ability to urinate spontaneously.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation of Rat Bone Marrow Stromal Cells (BMSCs) and characterization\u003c/h2\u003e \u003cp\u003eBone marrow stromal cells from rats were isolated following a previously described protocol. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The tibia and femur bones were removed, cleaned, and extracted from rats. The leg's skin, fur, and muscles were peeled off, and the bones were rinsed in 70% ethanol for one minute before being placed in a sterile PBS-filled petri dish. After this stage, all procedures were performed in a biological safety cabinet. The bone ends were cut with scissors, and the bone marrow was flushed into a 50ml tube with sterile PBS using a 22G needle attached to a 5ml syringe. This step was repeated two to three times for each bone. The cell suspension was passed through a 70 \u0026micro;m cell strainer to remove bone debris and blood aggregates. The isolated BMSCs were characterized according to a previously established protocol [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell culture\u003c/h2\u003e \u003cp\u003eTo prepare the bone marrow cells for culture, 10 ml of fresh DMEM medium (with 10% FBS and 1% pen-strep) was added to the extracted cells. The cells were then seeded in a 25cm2 flask and incubated in a 37℃ and 5% CO2 incubator for 3 hours. After 3 hours, the medium was replaced with a fresh DMEM medium. As the BMSCs can adhere to the surface of the culture flask, the suspension cells were excluded from this step. The culture medium was replaced with fresh medium every day for the first three days and then every 3 or 4 days. After approximately 2\u0026ndash;3 weeks, the cells had reached efficient confluences and could be subcultured when they reached a confluence of \u0026ge;\u0026thinsp;60%. The culture medium was removed, and the monolayer cells were rinsed with PBS. 2ml Trypsin-EDTA was added to the washed cells and incubated for 2\u0026ndash;5 minutes at 37℃ until the cells were detached. To inhibit trypsin action, 5\u0026ndash;10 ml media containing serum were added. The cells were collected in a tube and centrifuged for approximately 5 minutes at 400g. Then, the cell pellet was resuspended in fresh medium and dispensed into two flasks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Adenoviral vector\u003c/h2\u003e \u003cp\u003eFor this study, replication-defective adenoviruses containing bovine Fibromodulin (Ad5-FMOD), and adenoviruses containing the β-galactosidase gene (Ad5-LacZ) were kindly gifted by Dr. Paul Kingstone (The University of Manchester, U.K) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 BMSCs transduction by Ad5-FMOD vector\u003c/h2\u003e \u003cp\u003eTo determine the Adenoviral titration, a plaque assay was conducted in HEK 293 cells. Two days before transduction, BMSC cells were plated in a 24-well plate with 2 \u0026times; 105 cells per well. The Ad5-FMOD and Ad5-LacZ were diluted in RPMI 1640 serum-free culture media (Gibco, Invitrogen) and added to the cells with a multiplicity of infection (MOI) of 100 pfu/cell. The cells were then incubated at 37\u0026deg;C. Four hours after transduction, the media was removed, and the cells were washed with PBS and cultured again in fresh medium with 15% FBS for 48h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Evaluation of Fibromodulin expression by RT-PCR\u003c/h2\u003e \u003cp\u003eThe Qiagen RNeasy Mini Kit (Cat.No: 74104) was used for extracting total RNA from transduced cells, following the manufacturer's instructions. A total of 1 \u0026micro;g of RNA was utilized to generate cDNA, using the Qiagen One-Step RT-PCR cDNA Synthesis Kit as per the manufacturer's specifications. The RT-PCR was carried out with the following primers:\u003c/p\u003e \u003cp\u003eFMOD forward primer (FMF1) 5\u0026prime;-TGAAGGCAGCACCTGACCGC-3\u0026prime;, FMOD reverse primer (FMR1) 5\u0026prime;-ACGCCTTGGCTTCTCCTGCC-3\u0026prime; (189bp). β-Actin forward primer 5\u0026prime; AAGCAGGAGTATGACGAGTC-3\u0026prime;, β-Actin reverse primer 5\u0026prime; CCGTTCCAGTTTTTAAATCC-3\u0026prime; (207bp).\u003c/p\u003e \u003cp\u003eThe PCR was performed for 40 cycles under the following conditions: 30 min at 50\u0026deg;C, 15 min at 95\u0026deg;C, 45 sec at 94\u0026deg;C, 45 sec at 63\u0026deg;C, 1 min at 72\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Spinal Cord Injury Rat Model\u003c/h2\u003e \u003cp\u003eTo perform spinal cord injury, a \"weight dropping\" method was utilized. Thirteen rats were randomly assigned to each of the four groups. All rats were anesthetized by intraperitoneal injection of 87 mg of ketamine per kg of body weight and 10 mg of xylazine per kg of body weight. To prevent dryness of the cornea, a drop of mineral oil was used. The thoracic area was shaved and disinfected with povidone-iodine solution. Then, under a microscope, laminectomy surgery was performed on the tenth thoracic vertebra (T10). The spinal cord was injured by dropping a 10-gram metal rod from a 50-mm distance onto the exposed spinal cord (T10 vertebra). The wound was closed using chromic catgut (4/0) for the muscle and nylon suture (3/0) for the skin. After the surgery, rats were given Cefazolin (50 mg/kg BW/day intramuscular) for three days and placed on a 37\u0026deg;C heating blanket overnight [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Bone Marrow Stromal Cells Transplantation (BMSCT)\u003c/h2\u003e \u003cp\u003eFour days following a spinal cord contusion, 2x105 transduced cells were suspended in phosphate-buffered saline (PBS). Rats were then fixed in a stereotaxic device, and the cells were injected using a 5\u0026micro;l Hamilton syringe to a depth of 1.5mm in the caudal border of the lesion site for 120 seconds. The first group of rats received normal saline, the second group was administered with BMSCs, the third group was given BMSCs infected with adenovirus expressing beta-galactosidase, and the fourth group received BMSCs infected with adenovirus expressing Fibromodulin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Behavioral analysis\u003c/h2\u003e \u003cp\u003eThe behavioral functions of the animals were evaluated one week after cell injection. The locomotor BBB test was used to assess their progress for six weeks after surgery. The BBB test is an open field score that ranges from complete hind limb paralysis (Zero) to normal movement (Twenty-one). Two observers, who were unaware of the treatment, scored the animals according to the BBB scale [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 BDA anterograde CST tracing\u003c/h2\u003e \u003cp\u003eTo trace the corticospinal tract (CST), anterograde tracing was performed using BDA, which was administered as per standard procedures. The experiment involved 12 rats, with three rats randomly selected from each group. The rats were anesthetized, and their skulls were secured using a stereotaxic device. The surgical area was cleansed and sanitized with an iodine swab. A craniotomy was then performed, creating a hole approximately 1.0 mm in diameter and depth, at a location 2 mm lateral and 1.6 mm caudal to the bregma. Using a 5\u0026micro;l Hamilton syringe, a slow injection of 1 \u0026micro;l of 10% BDA (Life Technologies, Cat N: D-1956) was administered into the cerebral motor cortex at a depth of 1.5 mm for 160 seconds. The BDA was injected two weeks before the rats were sacrificed [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Histological procedures\u003c/h2\u003e \u003cp\u003eTo prepare for analysis, one centimeter of the spinal cord with the lesion at the midpoint was cut and embedded in paraffin. The embedded spinal cords were cut into serial transverse sections, each 5-\u0026micro;m thick with a 200 \u0026micro;m interval, using a freezing microtome (Rotary Microtome, YD-2508). BDA labeling was performed as previously described. Briefly, the sections were rinsed in 0.1M Tris-buffered saline (TBS; pH 7.4) or PBS and treated with 0.6% hydrogen peroxide in TBS or PBS for 30 minutes to inhibit endogenous peroxidase activity. They were then incubated with avidin-biotin-peroxidase complex (VECTASTAIN\u0026reg; Elite\u0026reg; ABC HRP Kit; PK-6100, USA). After washing the sections, they were treated in diaminobenzidine tetrahydrochloride (DAB) and nickel chloride until the production of a dark reaction. Sections were photographed under a Nikon microscope (\u0026times;40). The extent of the DAB labeled fibers in each section was quantified in a blinded manner using Scion Image software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS v16 (Chicago, Inc., USA) and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Statistical analysis involved one-way and two-way ANOVA, followed by post-hoc analysis with the Tukey test.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Transferred gene expression in vitro\u003c/h2\u003e \u003cp\u003eTo assess the expression level of Fibromodulin mRNA in vitro, reverse transcription-PCR (RT-PCR) was performed. The results showed that Fibromodulin mRNA expression was confirmed in the fourth group, as compared to the control groups (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The biological activity of an Adenoviral vector carrying the Fibromodulin gene was confirmed via a bioassay of TGF-β activity, as previously reported by P Ranjzad et al.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Recovery of hind limb function\u003c/h2\u003e \u003cp\u003eThe behavioral analysis started one week after injecting cells and continued weekly for 6 weeks after spinal cord injury (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To determine the locomotor recovery of rats, the BBB locomotion score was used, which considers the early (BBB score from 0 to 7), intermediate (8\u0026ndash;13) and late phases (14\u0026ndash;21) of recovery [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. According to statistical analysis, there were significant differences between groups during the 3rd, 5th, and 6th weeks. The probability values (p-values) were p\u0026thinsp;=\u0026thinsp;0.002, p\u0026thinsp;=\u0026thinsp;0.047, and p\u0026thinsp;=\u0026thinsp;0.006, respectively. However, no differences in BBB scores were found between all groups at other time points (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant differences were observed until 1 week after injury between groups (p-value\u0026thinsp;=\u0026thinsp;0.325). In the third week, the first signs of locomotor function recovery were observed. There were significant differences between the fourth and control groups (p-value\u0026thinsp;=\u0026thinsp;0.00). At week six, the average score for the control group (group 1) was 5.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.140, for the second group was 8.00, for the third group was 8.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.707, and for the fourth group was 9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.675. The average BBB score for the fourth group was significantly higher than the control group (p-value\u0026thinsp;=\u0026thinsp;0.03) over six weeks, indicating that the Fibromodulin gene had a significant effect on functional recovery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Quantification of CST axons\u003c/h2\u003e \u003cp\u003eData collected from BDA anterograde tracing of CST fibers were analyzed to examine the diffusion pattern of sprouting CST fibers after spinal cord injury. The lowest axon count was obtained in the control group, with an average of 87.07\u0026thinsp;\u0026plusmn;\u0026thinsp;46.75. The average number of axons was 466.33\u0026thinsp;\u0026plusmn;\u0026thinsp;146.959 in group 2, 474.13\u0026thinsp;\u0026plusmn;\u0026thinsp;109.149 in group 3, and 829.40\u0026thinsp;\u0026plusmn;\u0026thinsp;139.006 in group 4. Groups 2, 3, and 4 showed significantly higher numbers of axons compared to the control group (P\u0026thinsp;\u0026le;\u0026thinsp;0.001). The number of axons in group 4 was statistically significant versus groups 2 and 3 (P\u0026thinsp;\u0026le;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eA spinal cord injury (SCI) is a lesion on any part of the spinal cord that leads to short-term or steady-state changes in its normal motor, sensory, or autonomic function. Unfortunately, damaged axons do not generally regenerate and so far, there is no efficient clinically approved strategy to cure SCI [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. FMOD, a member of the SLRP family, is recognized for its interaction with collagen fibrils and the configuration of the extracellular matrix. Previous studies have reported that FMOD plays a significant role in cell fate determination, fetal-type scarless wound healing stimulates adult wound closure, and decreases scar formation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent years have seen stem cell transplantation, such as bone marrow mesenchymal cells (BMSCs), embryonic stem cells (ESCs), and umbilical cord blood stem cells, being used to treat spinal cord injury (SCI). This new strategy has shown promise in activating neuroregeneration and restoring spinal cord functions [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. BMSCs, in particular, have demonstrated good differentiation potential and neural recovery. They are capable of differentiating into glial cells and neurons, repairing the myelin sheath of injured axons, and regenerating nerve fibers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Additionally, BMSCs produce various trophic factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF), and cytokines such as IL-6 and stem cell factor (SCF), and IGF-1, which are effective in promoting neural protection or regeneration [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Transplanted BMSCs produce neurotrophic factors such as BDNF and GDNF that can reduce neuronal cell death protect injured neural tissue and promote axon regrowth, respectively [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These factors make BMSC transplantation a promising method for treating SCI. In 2000, Chopp and colleagues demonstrated motor improvement using BMSCs transplantation at the site of the injury in rat models [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In 2003, Wu and colleagues investigated bone marrow stromal cell grafting in the lesion site. Transplanted BMSCs stimulated the regeneration of the injured spinal cord by raising tissue repair of the lesion and resulted in smaller cavities than in controls. In another study, Ide and colleagues reduced cavity formation and myelinated injured axons, and increased the BBB score to 9.8 (compared to 5.5\u0026ndash;5.7 in the control group) using direct transplantation of BMSCs in the lesion site two weeks post-injury in subacute-spinal-cord injury [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. To investigate the therapeutic effects of Fibromodulin in combination with BMSCs in a rat spinal cord injury model, the present study transduced BMSCs with adenoviral vectors carrying the Fibromodulin gene. These modified cells were then transplanted into SCI, leading to promoting axonal regeneration and functional recovery. The three BMSCs transplanted groups (BMSCs, BMSCs -LacZ, and BMSCs -Fibromodulin) showed significant axonal regeneration when compared with rats in the control group. This is consistent with previous reports showing that BMSCs injection increases the capability of axon regrowth at the injury site [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], [\u003cspan additionalcitationids=\"CR58 CR59\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The study found that transplantation of Fibromodulin-expressing BMSCs into the spinal cord four days after injury significantly improved functional outcomes, as evaluated on the BBB test. Significant recovery of functional outcomes extended up to 6 weeks after transplantation. BBB locomotor scaling score results indicated significant scores in weeks 2, 3, 4, 5, and 6 after injection. Behavioral follow-up was performed 6 weeks post-injury. There was no statistical difference until 1 week after injury between groups (p value\u0026thinsp;=\u0026thinsp;0.325). The average score in the 6th week was 5.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.140, 8.00, 8.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.707, and 9.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.675 for groups 1st, 2nd, 3rd, and 4th, respectively. The fourth group had a statistically significant BBB score in comparison with the control group (p value\u0026thinsp;=\u0026thinsp;0.03) in the sixth week, indicating that the Fibromodulin gene had a significant effect on functional recovery. In the third week, the first signs of locomotor function recovery were observed, and they were statistically significant between the fourth and control groups (p value\u0026thinsp;=\u0026thinsp;0.00).\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur study suggests that Fibromodulin may have a significant role in promoting axonal growth after severe injury. As there is no similar study to compare the results with, our focus was to investigate the potential of Fibromodulin for gene therapy of spinal cord injury (SCI) for the first time. Our study also confirmed the positive effect of bone marrow cell therapy combined with gene therapy. Therefore, our findings indicate that the combination of cell therapy (using BMSCs) and gene therapy (using Fibromodulin) can be considered a promising approach for gene therapy of SCI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAnimal Ethics \u0026quot;All experimental protocols were undertaken in compliance with the Institutional Animal Care \u0026amp; Use Committee (IACUC) standards and approved by the Zanjan University of Medical Sciences Ethics Review Board\u0026quot;.\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical guidelines set by the Ethics Committee of Zanjan University of Medical Sciences were strictly followed in caring for the animals.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. This manuscript does not include any individual person\u0026rsquo;s data in any form (including individual details, images, or videos).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Zanjan University of Medical Sciences [grant number 88112003]; and council for Stem cell Science and Technology [grant number 700/391] (Presidency of the Islamic Republic of Iran Vice-presidency for Science and Technology).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: MAK, AB, PR, PAK. Acquisition of data: MAK, MA, IJA. Analysis and interpretation of data: SM, MAK, MA, And AB. Drafting the article: MAK, MA. Critically revising the article: IJA, SM, PR, and PAK. Reviewed submitted version of manuscript: MAK, MA, IJA, SM, PR, PAK, and AB.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Zanjan University of Medical Sciences, we also wish to thank Prof. Paul Kingston and Parisa Ranjzad, Vascular Gene Therapy Unit, Research School of Clinical \u0026amp; Laboratory Sciences, Manchester Academic Health Science Centre, The University of Manchester, Manchester, UK, for giving us adenoviral vectors.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eM. G. F. Christopher D. Witiw, \u0026ldquo;Acute Spinal Cord Injury,\u0026rdquo; \u003cem\u003eJ. Spinal Disord. Tech.\u003c/em\u003e, vol. 28, no. 6, pp. 202\u0026ndash;210, Jul. 2015, doi: 10.1097/bsd.0000000000000287.\u003c/li\u003e\n\u003cli\u003eJ. Bennett, J. M. Das, and P. D. Emmady, \u0026ldquo;Spinal Cord Injuries,\u0026rdquo; \u003cem\u003eStatPearls\u003c/em\u003e, May 2022.\u003c/li\u003e\n\u003cli\u003eNational Spinal Cord Injury Statistical Center, \u0026ldquo;Spinal cord injury facts and figures at a glance.,\u0026rdquo; \u003cem\u003eJ. Spinal Cord Med.\u003c/em\u003e, vol. 36, no. 1, pp. 1\u0026ndash;2, 2013, doi: 10.1179/1079026813Z.000000000136.\u003c/li\u003e\n\u003cli\u003eA. Singh, L. Tetreault, S. Kalsi-Ryan, A. Nouri, and M. G. Fehlings, \u0026ldquo;Global prevalence and incidence of traumatic spinal cord injury.,\u0026rdquo; \u003cem\u003eClin. Epidemiol.\u003c/em\u003e, vol. 6, pp. 309\u0026ndash;31, 2014, doi: 10.2147/CLEP.S68889.\u003c/li\u003e\n\u003cli\u003eS. B. Jazayeri \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Prevalence of Spinal Cord Injury in Iran: A 3-Source Capture-Recapture Study,\u0026rdquo; \u003cem\u003eNeuroepidemiology\u003c/em\u003e, vol. 45, no. 1, pp. 28\u0026ndash;33, Aug. 2015, doi: 10.1159/000435785.\u003c/li\u003e\n\u003cli\u003eS. Thuret, L. D. F Moon, and F. H. Gage, \u0026ldquo;Autonomic dysreflexia Therapeutic interventions after spinal cord injury,\u0026rdquo; 2006, doi: 10.1038/nrn1955.\u003c/li\u003e\n\u003cli\u003eR. Lima, A. Monteiro, A. J. Salgado, S. Monteiro, and N. A. Silva, \u0026ldquo;Pathophysiology and Therapeutic Approaches for Spinal Cord Injury,\u0026rdquo; \u003cem\u003eInt. J. Mol. Sci. 2022, Vol. 23, Page 13833\u003c/em\u003e, vol. 23, no. 22, p. 13833, Nov. 2022, doi: 10.3390/IJMS232213833.\u003c/li\u003e\n\u003cli\u003eA. Anjum \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Spinal Cord Injury: Pathophysiology, Multimolecular Interactions, and Underlying Recovery Mechanisms,\u0026rdquo; \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e, vol. 21, no. 20, pp. 1\u0026ndash;35, Oct. 2020, doi: 10.3390/IJMS21207533.\u003c/li\u003e\n\u003cli\u003eR. Stewart and S. Przyborski, \u0026ldquo;Non-neural adult stem cells: tools for brain repair?,\u0026rdquo; \u003cem\u003eBioEssays\u003c/em\u003e, vol. 24, no. 8, pp. 708\u0026ndash;713, Aug. 2002, doi: 10.1002/bies.10124.\u003c/li\u003e\n\u003cli\u003eC. S. Padovan \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Expression of neuronal markers in differentiated marrow stromal cells and CD133+ stem-like cells.,\u0026rdquo; \u003cem\u003eCell Transplant.\u003c/em\u003e, vol. 12, no. 8, pp. 839\u0026ndash;48, 2003.\u003c/li\u003e\n\u003cli\u003eC. Lv, T. Zhang, K. Li, and K. Gao, \u0026ldquo;Bone marrow mesenchymal stem cells improve spinal function of spinal cord injury in rats via TGF-\u0026beta;/Smads signaling pathway,\u0026rdquo; \u003cem\u003eExp. Ther. Med.\u003c/em\u003e, 2020, doi: 10.3892/etm.2020.8640.\u003c/li\u003e\n\u003cli\u003eS. Bai, H. Zhou, and L. Wu, \u0026ldquo;Bone marrow stromal cells improved functional recovery in spinal cord injury rats partly via the Toll-like receptor-4/nuclear factor-\u0026kappa;B signaling pathway,\u0026rdquo; \u003cem\u003eExp. Ther. Med.\u003c/em\u003e, vol. 17, no. 1, p. 444, Oct. 2019, doi: 10.3892/ETM.2018.6907.\u003c/li\u003e\n\u003cli\u003eO. Honmou \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Intravenous infusion of auto serum-expanded autologous mesenchymal stem cells in spinal cord injury patients: 13 case series,\u0026rdquo; \u003cem\u003eClin. Neurol. Neurosurg.\u003c/em\u003e, vol. 203, p. 106565, Apr. 2021, doi: 10.1016/J.CLINEURO.2021.106565.\u003c/li\u003e\n\u003cli\u003eJ.-B. Lee \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;A pre-clinical assessment model of rat autogeneic bone marrow stromal cell transplantation into the central nervous system.,\u0026rdquo; \u003cem\u003eBrain Res. Brain Res. Protoc.\u003c/em\u003e, vol. 14, no. 1, pp. 37\u0026ndash;44, Nov. 2004, doi: 10.1016/j.brainresprot.2004.09.004.\u003c/li\u003e\n\u003cli\u003eG. Liu, Y. Wu, F. Kong, S. Ma, L. Fu, and J. Geng, \u0026ldquo;BMSCs differentiated into neurons, astrocytes and oligodendrocytes alleviated the inflammation and demyelination of EAE mice models,\u0026rdquo; \u003cem\u003ebioRxiv\u003c/em\u003e, p. 2020.11.16.384354, Nov. 2020, doi: 10.1101/2020.11.16.384354.\u003c/li\u003e\n\u003cli\u003eH. Cheng \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Cyclic Strain and Electrical Co-stimulation Improve Neural Differentiation of Marrow-Derived Mesenchymal Stem Cells,\u0026rdquo; \u003cem\u003eFront. Cell Dev. Biol.\u003c/em\u003e, vol. 9, p. 624755, May 2021, doi: 10.3389/FCELL.2021.624755/BIBTEX.\u003c/li\u003e\n\u003cli\u003eA. M. Parr, C. H. Tator, and A. Keating, \u0026ldquo;Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury,\u0026rdquo; \u003cem\u003eBone Marrow Transplant.\u003c/em\u003e, vol. 40, pp. 609\u0026ndash;619, 2007, doi: 10.1038/sj.bmt.1705757.\u003c/li\u003e\n\u003cli\u003eP. Bossolasco \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Neuro-glial differentiation of human bone marrow stem cells in vitro,\u0026rdquo; 2005, doi: 10.1016/j.expneurol.2004.12.013.\u003c/li\u003e\n\u003cli\u003eR. Io Garc Ia, J. Aguiar, E. Alberti, K. De La Cu Etara, and N. Pav, \u0026ldquo;Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors,\u0026rdquo; doi: 10.1016/j.bbrc.2004.02.111.\u003c/li\u003e\n\u003cli\u003eT. R. Brazelton, F. M. Rossi, G. I. Keshet, and H. M. Blau, \u0026ldquo;From marrow to brain: expression of neuronal phenotypes in adult mice.,\u0026rdquo; \u003cem\u003eScience\u003c/em\u003e, vol. 290, no. 5497, pp. 1775\u0026ndash;9, Dec. 2000.\u003c/li\u003e\n\u003cli\u003eS. Chakravarti, J. Paul, L. Roberts, I. Chervoneva, A. Oldberg, and D. E. Birk, \u0026ldquo;Ocular and scleral alterations in gene-targeted lumican-fibromodulin double-null mice,\u0026rdquo; \u003cem\u003eInvestig. Ophthalmol. Vis. Sci.\u003c/em\u003e, vol. 44, no. 6, pp. 2422\u0026ndash;2432, 2003, doi: 10.1167/iovs.02-0783.\u003c/li\u003e\n\u003cli\u003eK. Anden\u0026aelig;s \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;The extracellular matrix proteoglycan fibromodulin is upregulated in clinical and experimental heart failure and affects cardiac remodeling,\u0026rdquo; \u003cem\u003ePLoS One\u003c/em\u003e, vol. 13, no. 7, pp. 1\u0026ndash;20, 2018, doi: 10.1371/journal.pone.0201422.\u003c/li\u003e\n\u003cli\u003eF. Zhao, Y. Bai, X. Xiang, and X. Pang, \u0026ldquo;The role of fibromodulin in inflammatory responses and diseases associated with inflammation,\u0026rdquo; \u003cem\u003eFront. Immunol.\u003c/em\u003e, vol. 14, p. 1191787, Jul. 2023, doi: 10.3389/FIMMU.2023.1191787/BIBTEX.\u003c/li\u003e\n\u003cli\u003eZ. Zheng, H. S. Granado, and C. Li, \u0026ldquo;Fibromodulin, a Multifunctional Matricellular Modulator,\u0026rdquo; \u003cem\u003ehttps://doi.org/10.1177/00220345221138525\u003c/em\u003e, vol. 102, no. 2, pp. 125\u0026ndash;134, Dec. 2022, doi: 10.1177/00220345221138525.\u003c/li\u003e\n\u003cli\u003eZ. Zheng \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Fibromodulin reduces scar formation in adult cutaneous wounds by eliciting a fetal-like phenotype,\u0026rdquo; \u003cem\u003eSignal Transduct. Target. Ther.\u003c/em\u003e, vol. 2, p. 17050, Oct. 2017, doi: 10.1038/sigtrans.2017.50.\u003c/li\u003e\n\u003cli\u003eZ. Zheng \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Fibromodulin-Deficiency Alters Temporospatial Expression Patterns of Transforming Growth Factor-\u0026beta; Ligands and Receptors during Adult Mouse Skin Wound Healing,\u0026rdquo; \u003cem\u003ePLoS One\u003c/em\u003e, vol. 9, no. 3, p. e90817, Mar. 2014, doi: 10.1371/journal.pone.0090817.\u003c/li\u003e\n\u003cli\u003eA. Stoff \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Effect of adenoviral mediated overexpression of fibromodulin on human dermal fibroblasts and scar formation in full-thickness incisional wounds,\u0026rdquo; \u003cem\u003eJ. Mol. Med.\u003c/em\u003e, vol. 85, no. 5, pp. 481\u0026ndash;496, May 2007, doi: 10.1007/s00109-006-0148-z.\u003c/li\u003e\n\u003cli\u003eM. Kohta, E. Kohmura, and T. Yamashita, \u0026ldquo;Inhibition of TGF-b1 promotes functional recovery after spinal cord injury,\u0026rdquo; doi: 10.1016/j.neures.2009.08.017.\u003c/li\u003e\n\u003cli\u003eJ. C. Hsu \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Matrix metalloproteinase-2 facilitates wound healing events that promote functional recovery after spinal cord injury.,\u0026rdquo; \u003cem\u003eundefined\u003c/em\u003e, 2006.\u003c/li\u003e\n\u003cli\u003eM. Koda \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Adenovirus Vector-Mediated \u003cem\u003eIn Vivo\u003c/em\u003e Gene Transfer of Brain-Derived Neurotrophic Factor (BDNF) Promotes Rubrospinal Axonal Regeneration and Functional Recovery after Complete Transection of the Adult Rat Spinal Cord,\u0026rdquo; \u003cem\u003eJ. Neurotrauma\u003c/em\u003e, vol. 21, no. 3, pp. 329\u0026ndash;337, Mar. 2004, doi: 10.1089/089771504322972112.\u003c/li\u003e\n\u003cli\u003eY. Zhang \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Enhanced Adenoviral Gene Delivery to Motor and Dorsal Root Ganglion Neurons Following Injection Into Demyelinated Peripheral Nerves,\u0026rdquo; doi: 10.1002/jnr.22394.\u003c/li\u003e\n\u003cli\u003eY. Liu \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Application of recombinant adenovirus for in vivo gene delivery to spinal cord,\u0026rdquo; \u003cem\u003eBrain Res.\u003c/em\u003e, vol. 768, no. 1\u0026ndash;2, pp. 19\u0026ndash;29, Sep. 1997, doi: 10.1016/S0006-8993(97)00587-8.\u003c/li\u003e\n\u003cli\u003eA. P. Tosolini and R. Morris, \u0026ldquo;Viral-mediated gene therapy for spinal cord injury (SCI) from a translational neuroanatomical perspective.,\u0026rdquo; \u003cem\u003eNeural Regen. Res.\u003c/em\u003e, vol. 11, no. 5, pp. 743\u0026ndash;4, May 2016, doi: 10.4103/1673-5374.182698.\u003c/li\u003e\n\u003cli\u003eA. A. Abdellatif \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Gene delivery to the spinal cord: comparison between lentiviral, adenoviral, and retroviral vector delivery systems.,\u0026rdquo; \u003cem\u003eJ. Neurosci. Res.\u003c/em\u003e, vol. 84, no. 3, pp. 553\u0026ndash;67, Aug. 2006, doi: 10.1002/jnr.20968.\u003c/li\u003e\n\u003cli\u003eA. O. Sosnovtseva \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Recombinant Adenoviruses for Delivery of Therapeutics Following Spinal Cord Injury,\u0026rdquo; \u003cem\u003eFront. Pharmacol.\u003c/em\u003e, vol. 12, Jan. 2021, doi: 10.3389/FPHAR.2021.777628.\u003c/li\u003e\n\u003cli\u003eF. C. Zhu, D. M. Jiang, M. H. Zhang, B. Zhao, C. He, and J. Yang, \u0026ldquo;Adenovirus vector-mediated in vivo gene transfer of nuclear factor erythroid-2p45-related factor 2 promotes functional recovery following spinal cord contusion,\u0026rdquo; \u003cem\u003eMol. Med. Rep.\u003c/em\u003e, vol. 20, no. 5, pp. 4285\u0026ndash;4292, 2019, doi: 10.3892/mmr.2019.10687.\u003c/li\u003e\n\u003cli\u003eM. Soleimani and S. Nadri, \u0026ldquo;A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow,\u0026rdquo; \u003cem\u003eNat. Protoc.\u003c/em\u003e, vol. 4, no. 1, pp. 102\u0026ndash;106, Jan. 2009, doi: 10.1038/nprot.2008.221.\u003c/li\u003e\n\u003cli\u003eM. Shahrezaie \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Improved stem cell therapy of spinal cord injury using GDNF-overexpressed bone marrow stem cells in a rat model,\u0026rdquo; \u003cem\u003eBiologicals\u003c/em\u003e, vol. 50, pp. 73\u0026ndash;80, Nov. 2017, doi: 10.1016/j.biologicals.2017.08.009.\u003c/li\u003e\n\u003cli\u003eP. Ranjzad, H. K. Salem, and P. A. Kingston, \u0026ldquo;Adenovirus-mediated gene transfer of fibromodulin inhibits neointimal hyperplasia in an organ culture model of human saphenous vein graft disease,\u0026rdquo; \u003cem\u003eGene Ther.\u003c/em\u003e, vol. 16, no. 9, pp. 1154\u0026ndash;1162, Sep. 2009, doi: 10.1038/gt.2009.63.\u003c/li\u003e\n\u003cli\u003eB. T. Kalish and M. J. Whalen, \u0026ldquo;Weight Drop Models in Traumatic Brain Injury,\u0026rdquo; Humana Press, New York, NY, 2016, pp. 193\u0026ndash;209.\u003c/li\u003e\n\u003cli\u003eJ. A. GRUNER, \u0026ldquo;A Monitored Contusion Model of Spinal Cord Injury in the Rat,\u0026rdquo; \u003cem\u003eJ. Neurotrauma\u003c/em\u003e, vol. 9, no. 2, pp. 123\u0026ndash;128, Jan. 1992, doi: 10.1089/neu.1992.9.123.\u003c/li\u003e\n\u003cli\u003eY. Bomstein \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Features of skin-coincubated macrophages that promote recovery from spinal cord injury,\u0026rdquo; \u003cem\u003eJ. Neuroimmunol.\u003c/em\u003e, vol. 142, no. 1\u0026ndash;2, pp. 10\u0026ndash;16, Sep. 2003, doi: 10.1016/S0165-5728(03)00260-1.\u003c/li\u003e\n\u003cli\u003eT. E. P. de Barros Filho and A. E. I. S. Molina, \u0026ldquo;Analysis of the sensitivity and reproducibility of the Basso, Beattie, Bresnahan (BBB) scale in Wistar rats.,\u0026rdquo; \u003cem\u003eClinics (Sao Paulo).\u003c/em\u003e, vol. 63, no. 1, pp. 103\u0026ndash;8, Feb. 2008.\u003c/li\u003e\n\u003cli\u003eD. M. BASSO, M. S. BEATTIE, and J. C. BRESNAHAN, \u0026ldquo;A Sensitive and Reliable Locomotor Rating Scale for Open Field Testing in Rats,\u0026rdquo; \u003cem\u003eJ. Neurotrauma\u003c/em\u003e, vol. 12, no. 1, pp. 1\u0026ndash;21, Feb. 1995, doi: 10.1089/neu.1995.12.1.\u003c/li\u003e\n\u003cli\u003eI. A. Ferguson, C. Xian, E. Barati, and R. A. Rush, \u0026ldquo;Comparison of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury.,\u0026rdquo; \u003cem\u003eJ. Neurosci. Methods\u003c/em\u003e, vol. 109, no. 2, pp. 81\u0026ndash;9, Aug. 2001.\u003c/li\u003e\n\u003cli\u003eS. M. Willerth and S. E. Sakiyama-Elbert, \u0026ldquo;Cell therapy for spinal cord regeneration.,\u0026rdquo; \u003cem\u003eAdv. Drug Deliv. Rev.\u003c/em\u003e, vol. 60, no. 2, pp. 263\u0026ndash;76, Jan. 2008, doi: 10.1016/j.addr.2007.08.028.\u003c/li\u003e\n\u003cli\u003eZ. Zheng \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Fibromodulin Is Essential for Fetal-Type Scarless Cutaneous Wound Healing.,\u0026rdquo; \u003cem\u003eAm. J. Pathol.\u003c/em\u003e, vol. 186, no. 11, pp. 2824\u0026ndash;2832, 2016, doi: 10.1016/j.ajpath.2016.07.023.\u003c/li\u003e\n\u003cli\u003eJ. Jian \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Fibromodulin promoted in vitro and in vivo angiogenesis,\u0026rdquo; \u003cem\u003eBiochem. Biophys. Res. Commun.\u003c/em\u003e, vol. 436, no. 3, pp. 530\u0026ndash;535, Jul. 2013, doi: 10.1016/J.BBRC.2013.06.005.\u003c/li\u003e\n\u003cli\u003eZ. Zheng \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Fibromodulin Enhances Angiogenesis during Cutaneous Wound Healing,\u0026rdquo; \u003cem\u003ePlast. Reconstr. Surg. Glob. Open\u003c/em\u003e, vol. 2, no. 12, p. e275, Dec. 2014, doi: 10.1097/GOX.0000000000000243.\u003c/li\u003e\n\u003cli\u003eR. Vawda, J. Wilcox, and M. Fehlings, \u0026ldquo;Current stem cell treatments for spinal cord injury.,\u0026rdquo; \u003cem\u003eIndian J. Orthop.\u003c/em\u003e, vol. 46, no. 1, pp. 10\u0026ndash;8, Jan. 2012, doi: 10.4103/0019-5413.91629.\u003c/li\u003e\n\u003cli\u003eA. J. Mothe and C. H. Tator, \u0026ldquo;Advances in stem cell therapy for spinal cord injury.,\u0026rdquo; \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e, vol. 122, no. 11, pp. 3824\u0026ndash;34, Nov. 2012, doi: 10.1172/JCI64124.\u003c/li\u003e\n\u003cli\u003eM. Sasaki, O. Honmou, Y. Akiyama, T. Uede, K. Hashi, and J. D. Kocsis, \u0026ldquo;Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons,\u0026rdquo; \u003cem\u003eGlia\u003c/em\u003e, vol. 35, no. 1, pp. 26\u0026ndash;34, 2001, doi: 10.1002/glia.1067.\u003c/li\u003e\n\u003cli\u003eC. Ide \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat,\u0026rdquo; \u003cem\u003eBrain Res.\u003c/em\u003e, vol. 1332, pp. 32\u0026ndash;47, May 2010, doi: 10.1016/J.BRAINRES.2010.03.043.\u003c/li\u003e\n\u003cli\u003eM. Enomoto, \u0026ldquo;The future of bone marrow stromal cell transplantation for the treatment of spinal cord injury,\u0026rdquo; \u003cem\u003eNeural Regen. Res.\u003c/em\u003e, vol. 10, no. 3, p. 383, Mar. 2015, doi: 10.4103/1673-5374.153684.\u003c/li\u003e\n\u003cli\u003eM. Chopp \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Spinal cord injury in rat: Treatment with bone marrow stromal cell transplantation,\u0026rdquo; \u003cem\u003eNeuroreport\u003c/em\u003e, vol. 11, no. 13, pp. 3001\u0026ndash;3005, 2000, doi: 10.1097/00001756-200009110-00035.\u003c/li\u003e\n\u003cli\u003eS. Wu \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord,\u0026rdquo; \u003cem\u003eJ. Neurosci. Res.\u003c/em\u003e, vol. 72, no. 3, pp. 343\u0026ndash;351, May 2003, doi: 10.1002/jnr.10587.\u003c/li\u003e\n\u003cli\u003eJ. W. Kim, K. Y. Ha, J. N. Molon, and Y. H. Kim, \u0026ldquo;Bone marrow-derived mesenchymal stem cell transplantation for chronic spinal cord injury in rats: Comparative study between intralesional and intravenous transplantation,\u0026rdquo; \u003cem\u003eSpine (Phila. Pa. 1976).\u003c/em\u003e, vol. 38, no. 17, pp. 1065\u0026ndash;1074, 2013, doi: 10.1097/BRS.0b013e31829839fa.\u003c/li\u003e\n\u003cli\u003eC. Ide \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Bone marrow stromal cell transplantation for treatment of sub-acute spinal cord injury in the rat,\u0026rdquo; \u003cem\u003eBrain Res.\u003c/em\u003e, vol. 1332, pp. 32\u0026ndash;47, May 2010, doi: 10.1016/j.brainres.2010.03.043.\u003c/li\u003e\n\u003cli\u003eN. Nakano \u003cem\u003eet al.\u003c/em\u003e, \u0026ldquo;Effects of Bone Marrow Stromal Cell Transplantation through CSF on the Subacute and Chronic Spinal Cord Injury in Rats,\u0026rdquo; \u003cem\u003ePLoS One\u003c/em\u003e, vol. 8, no. 9, p. e73494, Sep. 2013, doi: 10.1371/journal.pone.0073494.\u003c/li\u003e\n\u003cli\u003eL. Lin, H. Lin, S. Bai, L. Zheng, and X. Zhang, \u0026ldquo;Bone marrow mesenchymal stem cells (BMSCs) improved functional recovery of spinal cord injury partly by promoting axonal regeneration,\u0026rdquo; \u003cem\u003eNeurochem. Int.\u003c/em\u003e, vol. 115, pp. 80\u0026ndash;84, May 2018, doi: 10.1016/J.NEUINT.2018.02.007.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fibromodulin, Bone marrow stromal cells, Gene therapy, Cell therapy, Spinal cord injury","lastPublishedDoi":"10.21203/rs.3.rs-5725598/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5725598/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpinal cord injury (SCI) can lead to various pathological changes which create an inappropriate environment for repair. The most important of such changes are glial scar and inhibition of neuronal growth in the injured site. Exogenous administration of genes that enhance neuronal survival, synaptic plasticity, and neurotransmission has been considered as a potential approach for treating SCI. Fibromodulin is one of those genes which can decrease TGF-β1 and increase MMP-2 expression, and consequently leads to a reduction in the glial scar, promotes the growth of axons, macrophage activation, and elimination of physical and molecular barriers of neuronal growth that will end with improvement in motor performance. Moreover, bone marrow stromal cells (BMSCs) can be a promising therapeutic strategy for SCI because they can secrete neural growth factors as well as differentiate into neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe randomly divided rats into four groups, each consisting of thirteen rats. The first group was administered normal saline, the second group received BMSCs, the third group received BMSCs infected with a beta-galactosidase expressing adenoviral vector, and the fourth group received BMSCs infected with a Fibromodulin expressing adenoviral vector. After inducing spinal cord injury using the weight-dropping method under general anesthesia, BMSCs were injected on the fourth-day post-injury. A Basso, Beattie, and Bresnahan (BBB) score test was conducted for six weeks. At the end of the fourth week, biotin dextran amine (BDA) was intracerebrally injected, and tissue staining was carried out two weeks after the injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe BBB locomotor score test was applied for six weeks. There were significant differences in BBB locomotor scale between the first and the fourthgroups. The mean score of the first group in the sixth week was 5.60, while it was 9.60 for the fourth group. There were significant differences in axon counting between the groups (P\u0026lt;0.000). The average number of axons counted from the first to the fourth group was 87.07, 466.33, 474.13, and 829.40, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsequently, our results highlight the therapeutic potential of the Fibromodulin expressing BMSCs for treating SCI.\u003c/p\u003e","manuscriptTitle":"Gene therapy of spinal cord injury using gene-modified Bone Marrow Stromal Cells with Fibromodulin expressing adenoviral vector in a rat SCI model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 04:28:39","doi":"10.21203/rs.3.rs-5725598/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"92721f51-a0f9-409e-ae91-55b0db493f9f","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-16T04:28:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-16 04:28:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5725598","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5725598","identity":"rs-5725598","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}