Pathophysiology, mechanisms and applications of mesenchymal stem cells for the treatment of spinal cord injury

Pathophysiology, mechanisms and applications of mesenchymal stem cells for the treatment of spinal cord injury

Biomedicine & Pharmacotherapy 91 (2017) 693–706 Available online at ScienceDirect www.sciencedirect.com Review Pathophysiology, mechanisms and app...

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Biomedicine & Pharmacotherapy 91 (2017) 693–706

Available online at

ScienceDirect www.sciencedirect.com

Review

Pathophysiology, mechanisms and applications of mesenchymal stem cells for the treatment of spinal cord injury Pravin Shende* , Muna Subedi Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’S NMIMS, Mumbai, India

A R T I C L E I N F O

Article history: Received 6 March 2017 Received in revised form 25 April 2017 Accepted 30 April 2017 Keywords: Spinal cord injury Transplantation Stem cells Bone marrow Mesenchymal cell

A B S T R A C T

Spinal Cord Injury (SCI) is a serious devastating condition associated to the high chances of morbidity and mortality. It involves a primary and a secondary injury, former cause damages to both lower and upper motor neurones and disrupts sensory, motor and autonomic functions while the latter involves various stages of molecular plus cellular incidents which elaborate the original injury. In the treatment of SCI, stem cells possess a good therapeutic potential. Bone marrow, adipose tissue, placenta, amniotic fluid and umbilical cord are the good sources for mesenchymal stem cells. This review article shows the uses of bone marrow derived mesenchymal cells in the treatment of acute and chronic case of SCI and its future scope. © 2017 Elsevier Masson SAS. All rights reserved.

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology of spinal cord injury . . . . . . Stem cell based therapy . . . . . . . . . . . . . . . . . Bone marrow mesenchymal stem cells . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . Financial and competing interests’ disclosure Conflict of interest . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Spinal cord injury (SCI) is well-defined as an injury or lesion that results due to the dysfunction imposed on the spinal cord thereby compromising the major functions of spinal cord viz; sensory, motor, autonomic, and reflex, either completely or partially due to trauma or disease or degeneration (non-trauma) [1,2]. Global incidence for SCI is estimated to be in 40–80 persons in a million population. Amongst these, 90% occurs due to trauma but the occurrence appears to be growing recently for the nontraumatic SCI [3]. The incidence of SCI level is shown in Fig. 1. Cervical region accounts for about 55% of acute SCI, and the rest

* Corresponding author at: Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’S NMIMS, Vile Parle (W), Mumbai, India. E-mail address: [email protected] (P. Shende). http://dx.doi.org/10.1016/j.biopha.2017.04.126 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

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three regions i.e. thoracic, thoracolumbar and lumbosacral, each report for about 15% of SCI [4]. The prevalence of SCI is about 54 cases per million population, as per census data in USA, thereby indicating about 17,000 new cases being re-counted each year. Male accounts for 80% of these incidences. When compared to cases of 1970s, the age for incidence has increased from then 29 years to 42 years now, however, the length of stay in hospital has decreased from 24 days to 11 days. When neurological category at the time of discharge of SCI patients is taken into consideration since 2010, 45% accounts for incomplete tetraplegia followed by 21.3%, 20% and 13.3% accounting for incomplete paraplegia, complete paraplegia and incomplete paraplegia respectively. Only 0.4% of SCI cases experience complete recovery on discharge from hospital [5]. SCI is the most serious complication that usually lead to neuronal death and axonal damage resulting in dyskinesia or somatosensory loss [6]. SCI interrupts the nerve connections

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Fig. 1. Level of Injury in Adult SCI.

between the brain and the body, and results in paralysis. The pathology of SCI is determined by both the primary mechanical injury and the secondary processes that prevails around hours and days after injury, includes ischemia, anoxia, inflammation, cavity and glial scar formations [7]. Spontaneous regeneration of neural tissue and the efficacy of therapies used for regeneration of axons is also compromised during secondary tissue damages of SCI [8]. Usually, the spinal cord axonal regeneration is subsidized by various factors, some of which includes diminution on inherent growth potential of CNS neurons, damaged CNS myelin that generate inhibitory signals, local astrocytes in reaction to the external stimuli forming glial scars and the absence of nerve growth factors and neurotrophic factors [9]. SCI is a serious damaging condition where patient experience significant sensory and functional loss, along with financial, social and emotional problems. SCI patients are at bigger risk of cardiovascular complexities, deep venous thrombosis, bed sores, osteoporosis, neuropathic pain and autonomic dysreflexia. SCI involves various complexities involved in its mechanism along with the failure on

Fig. 2. Mechanisms of damage after SCI in the cellular level.

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repairment and regeneration of neurons in the human body that limits the treatment of SCI and thus, makes it a multi-fronted challenge [10]. In this paper, we are presenting our recent understandings on SCI, pathophysiology associated to it and the applications of mesenchymal stem cells (MSCs) therapy in the treatment of SCI. Basic and clinical researchers associated in academics, business and various regulatory organizations who are interested or involved in stem cell research could be benefitted from this review paper. 2. Pathophysiology of spinal cord injury Primary injury and secondary injury are two categories of SCI. Primary injury, also known as physical injury, affects upper and lower motor neurones, and thus, has a range of adverse effects on cardiac outputs, vascular tones, respiration and sensory functions while the various molecular and cellular events that arise during secondary injury deepen the original injury [11]. Deepened injury creates an inhibitory environment which acts adverse to endogenous repairment, regeneration and remyelination efforts. Various secondary events such as inflammation, ischemia, oxidation of lipids and proteins, free radicals production, axonal degeneration, astrogliosis, necrosis and cellular apoptosis, as shown in Fig. 2 [12,13] occurs during secondary injury. The primary and secondary mechanisms of acute SCI is summarized in Fig. 3. Primary injury mechanisms include events like acute compression, laceration and shear at the site of injury, while the secondary injury mechanisms include majority of the changes or effects, in the systemic level which includes, changes in systemic effects, local vascular changes, electrolyte changes and biochemical changes that includes mostly neurotransmitter accumulation, along with edema, apoptosis and loss of

695

neurotrophic factor support. Some of these effects are briefly summarized in Fig. 4. On the basis of gross anatomic findings, human SCI can be categorized into four types as shown below in Fig. 5 [14]. Various histological changes that occurs during SCI as per different phases of SCI [14], are listed in Fig. 6. During SCI secondary phase, endogenous regeneration occurs i.e. the cells engage in repair and regeneration process, to minimize the lesion depth (via astrogliosis), to provide proper supply of blood forming new blood vessels, to cleanout cellular remains, to remodel and reunite the damaged neurons. Besides, secondary phase of SCI also serves as an available target site for therapeutic mediation of SCI, and for this, the most favourable and promising therapy is utilizing stem cells for the therapy [7,10]. 3. Stem cell based therapy Stem cell based therapy utilizes stem cells for treating or preventing a disease or condition. Stem cells (SC) are living cells that exists virtually in all multicellular beings. These can split and discern into various types of cell and renew themselves to generate further stem cells. In the body, different cell types can develop from these cells [15]. The differentiation mechanism of stem cells in a human body is shown in Fig. 7 as the human development continues. SC can be sourced from embryo, tissue, umbilical cord, and bone marrow depending upon the stage of development of human cell. Usually, in human embryonic SC (from inner cell mass of blastocysts) and adult SC (tissues) subsist. Normally, adult stem cells can be extracted from blood, bone marrow and adipose tissue. Three important characters of stem cells are ability of:  Renewing continuously

Fig. 3. Acute SCI mechanisms.

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Fig. 4. Various effects noticed throughout secondary mechanisms of acute SCI.

 Differentiating into somatic cell types, and  Limiting their own populace to a modest number [16]. A differentiating potential of a stem cell, which is also known as potency, gives us an indication of its characteristics such as the probability to discern into different types of cell. Potency of SC is the ability to separate into specific cell type and further can develop into a mature cell type [17]. Various potency of SC are:  Totipotency: can give rise to a complete and viable organism  Pluripotency: can discern into any tissue type, but in no case to a complete functioning living being  Multipotency: can discern into a few cells, but strictly to the only related cells type  Oligopotency: can discern into a limited cells type, e.g. lymphoidal or myeloidal cell types  Unipotency: can differentiate into a single cell type that can get renewal on itself [15,17]. SC are unique from other human body cells only at the DNA level, where gene expression is adaptable to signals inducing protein expression. Not only the paralysis after spinal cord injury, many serious diseases like Parkinson’s and Alzheimer’s are also

treatable with SC therapy. Recently in a research paper, it was found that after stem cells injection in a paralyzed spine cord injured mice showed improvement on their leg movements [18]. Categories of stem cells (SC) are:     

Embryonic SC Foetal SC Cord blood SC Adult SC Induced pluripotent SC [19]

Mesenchymal stem cells (MSCs) are multipotent prototype or stromal cells having multilineage potential that can discern into cells like adipocytes (fat cells), myocytes (muscle cells), osteocytes (bone cells) and chondrocytes (cartilage cells). Sources of MSCs can be adipose tissue, placenta, amniotic fluid, umbilical cord including bone marrow for easy isolation [20]. Reasons for favouring MSCs in SC therapy to treat SCI includes:  Easy for isolation and cryopreservation  Possess viability upholding and regenerative capability after isolation and cryopreservation at 80  C

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Fig. 5. Types of SCI based on gross anatomy.

Fig. 6. Histological changes during SCI.

 Good replication capabilities of healthy prototype cells with numerous differentiating possibility and  Negligible to no immunoreactivity or graft-versus-host response after allogeneic MSCs transplantation.

4. Bone marrow mesenchymal stem cells Bone marrow stromal cells can be sorted into two types:  Hematopoietic stem cells (HSCs) that separate into hematopoietic cell lines  Mesenchymal stem cells (MSCs) that separate into mesenchymal cell lines

Since HSCs and MSCs can be easily and reproducibly isolated from aspirates of bone marrow, and also can be easily grafted into the patients, these serves as great source for clinical transplantation. However, in terms of easy isolation, MSCs dominate the HSCs extraction [21]. Delivery method of cells is very crucial in treating SCI since direct injections in the injured tissue may amplify damage. The need of direct injection at the sites can be avoided with the use of MSCs as these possess tropism for damaged tissue sites. Recent researches have shown that labelled MSCs when given by various routes such as intraveneous, intrathecal, intralesional and intraspinal, in SCI and brain trauma rodents, have improved the conditions of these animal models since the labelled MSCs could migrate towards damaged CNS and even integrate up to 3 months of therapy. All the literatures used for references below (Tables 1 and 2) suggest that all kinds of SCI injury can be treated with

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Fig. 7. Stem cells differentiation in Human.

transplantation of MSCs. Mechanism of action behind the therapy solely depends as per the injury types on the molecular level [22– 25], however, bone marrow derived MSCs can be of promising therapeutic potential for treating the SCI. In this review paper, we will be discussing regarding the applications of bone marrow derived mesenchymal cells in SCI, as obtained from clinical trials and research works done on SCI, which are shown in Tables 1 and 2 as below. Table 2 shows that the BMSCs i.e. bone marrow derived mesenchymal stem cells can be transplanted by giving injections via intrathecal, intramedullary, intravenous, or intra-arterial routes. It also has an added advantage of being autologous and low teratogenicity. Also, the use of autologous mesenchymal stem cells and bone marrow mesenchymal stem cells elicit to be a safe, feasible, and reliable method for cellular transplantation in treating SCI, and thus, can be the main source of transplant which can be applied in humans. In the animal models, it has been reported that haematopoietic stem cell cannot be a candidate for SCI treatment as the CD34 cells did not result in myelination, thereby enhancing the fact that only MSC can be potential candidate for SCI treatment, as it forms myelination and repair the injury [27]. Also, when the transplantation of MSCs was carried out after a gap of some period post SCI rather than immediately post SCI, former provided more beneficial results since the gap period enhances the survival of cell along with its potential effect on functional recovery [28]. Thus, chronic SCI is more beneficially treated with MSCs. Similarly, in MSCs, autologous bone marrow stromal cells which was acutely isolated proves to be an efficient and renewable source of cells for demyelinating disease like SCI, when compared to allogenic MSCs and controls [29,43]. In one literature, it has been reported that the case of severe paraplegic and chronic SCI is mostly promoted by the BMSC transplantation [33]. All these reports confirm that BMSCs is truly an effective source for transplantation in SCI.

BMSC treatment was found to be superior to dantrolene in improving the locomotory rate of injured recipients. Combination of anti-matrix metalloproteinases with stem cells is believed to be the result of MSC transplantation [47]. BMSCs serves as a supporter to the matrix stimulating intraspinal axonal growth thereby promoting functional recovery in SCI [50]. Homogenized human clonal BMSCs was found to be more effective than plain BMSCs in regeneration of axonal neuron around the injury, which could be due to the homogeneous nature and high proliferative nature of homogenized cells [51]. When cell transplantation method or route is taken into consideration, various routes have been reported in the literature. When cells are transplanted through CSF, via lumbar puncture with no severe invasion, it can add a benefit of having repetitive administration, and it can promote both tissue repairment and behavioural recovery. Also, the cavity in rodent models was filled in both mild and severe injury, but the size of cavity was reduced more in the severely injured SCI rats when compared to their respective controls [32]. When intravenous and intralesion method for BMSC transplantation is compared in rodents, intralesion method showed more positive effects in both the functional recovery and modification of mass of leg muscle, when compared to the intravenous method [40]. Likewise, lumbar puncturing is more effective method of transplantation than direct injection and intravenous route, as the host immune response is decreased and cell engraftment is more positive with lumbar puncturing [41]. Similarly, intraspinal administration of bone marrow cells can significantly improve on thoracic SCI models [46] and BMSCs transplantation is more effective with intralesional administration when the recipient has chronic SCI [48]. The clear mechanism of treating SCI is yet unclear, however, in general, when SCI occurs, fibroblasts migrate to the site of injury to proliferate and differentiate into myofibroblasts which secretes type IV collagen and fibronectin as an extracellular matrix, that

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Table 1 Various cases of SCI in animal prototypes and the effects seen after bone marrow stem cell transplantation. S. N.

Injury or Lesion

1

Donor: Rat Thoracic (T10 level): A lesion was created by dropping a Recipient: Rat weight of 10 g from 25 mm height with 2.5 impactor head.

2

Thoracic (T10 level): Focal demyelinated lesion was made with radiotherapy machine in the dorsal segment of the spine utilizing x-irradiation and ethidium bromide (EB) injection.

Donor: Adult LacZ transgenic mice Recipient: Wistar rats

3

Thoracic (T9 level): Laminectomy was done with the use of impact rod dropping it from 25 mm height, under halothane anaesthesia.

Donor: Male Lewis rats Recipient: Female Lewis rats

4

Thoracic (T10 level): A focal demyelinated lesion was created using radiotherapy machine employing xirradiation and EB injection.

Donor: GFP expressing Mice Recipient: Wistar rats

5

Mid-thoracic (T8–T9 level): Lamina was removed to create lesion using a metal rod of 10 g weight that was released from 50 mm height.

6

Mid-thoracic (T8 level): Laminectomy was done at T8 level, dropping injury device onto the exposed part, weight dropped with 1 mm in spinal tissue to create injury. Mid-thoracic (T8–T9 level): Lamina was removed using a metal rod of 10 g and dropping from 12.5 mm height (mild injury) or 25 mm height (severe injury).

Donor: SpragueDawley rats Recipient: Sprague Dawley rats Donor: Male Wistar rats Recipient: Female Wistar rats

7

8

9

10

11

Donor/ Recipient species

Transplant

Histological signals

2.5  105 cells were given parenterally directly post 1 week of SCI.

Injured tissue showed even distribution of transplanted MSCs, and these possessed neuronal biomarker, i.e. NeuN.

Functional consequences

The motor functions of rats had improved substantially assessed by Basso, Beatie and Bresnahan (BBB) locomotor scale method. Remyelination of dorsal axonal Dorsal nerve bundles showed 1 104 CD34+ bone marrow stem cells or cultured MSCs bundles, diameter and number high myelination. was given parenterally at the of cell groups were observed No functional assessments site of injury after 3 days of EB and counted in rats. were seen. injection. Recipients were given cyclosporin A (10 mg/kg/day) for immunosuppression during transplantation. Significant improvement on Transplanted MSCs formed 3  105 cultured MSCs were directly injected into the lesion bridges across the epicentre of BBB scores was seen in immediately or 1 week of the lesion and these disclosed delayed implantation in injury. astrocytes and nerve fibers too. comparison to immediate NeuN and fibronectin-IR were treatment. expressed by MSC bundles. 5  103 cultured MSCs were Peripheral and central patterns Conduction velocity of intravenously injected after of remyelination had occurred remyelinated axons were 3 days of injury. in recipients. found to be increased, and CD44, collagen I and was significantly faster than fibronectin were coexpressed the demyelinated axons. on stromal cells. Cavity size had reduced due to Significant improvement on 1 106 cultured MSCs were given parenterally into the tissue repair at the injury site BBB scores was seen lesion directly after SCI. and the expression of collagen Remarkable coordination fibres at those sites. between hind limbs and forelimbs were seen after 3 weeks. Increased neuro-filament 3  105 cultured MSCs were No improvements on BBB slowly injected into the injury staining was observed along locomotory score. site after 2 days of SCI. with reduced cavity formation But increased spontaneous in lesion sites of MSC air stepping (coordinated transplantation. limb activity) was observed in transplanted animals. 6 5  10 of cultivated MSCs were BMSC injected rats showed BMSCs injection showed introduced into the 4th smaller cavity than the significant improvement of ventricle. controls, in both the types of BBB scores in recipients, injury (mild/severe) compared to controls. Cavity wall showed abundance of Glial Fibrillary Acidic Protein (GFAP)-positive astrocytes and neuronal elements.

Donor: SpragueDawley rats and Wistar rats Recipient: SpragueDawley rats for mild lesion and Wistar rats for severe lesion 1 106 of MSCs were Transplanted MSCs formed Donor: Male Mid-thoracic (T6–T8 level): Laminectomy was performed Wistar rat introduced in the lesion post 3 tissue bundles across the using weight drop device Recipient: months SCI. centromedullary injury and Female Wistar method where 12 mm2 decreased cavity too. rats MSCs expressed neuro filament cylinder of 25 g was dropped from a height of 20 cm. or GFAP. Cervical: a Kopf microwire Donor: Fischer 2  105 cultured MSCs, brainNeuronal MSCs = MSCs but < knife was used along the spinal 344 female derived neurotrophic factor BDNF-MSCs in providing dorsal midline to create a rats (BDNF)-MSCs, neurally neuronal differentiation (not at dorsal column lesion of 2 mm. Recipient: induced MSCs and neurally all) and axonal regeneration (to Fischer 344 induced BDNF-MSCs were some extent). female rats instantly inserted into the lesion post SCI. 5  105 cultured MSCs Donor: Transplanted cells occupied the Cervical (C3–C4 level): Healthy Laminectomy was made to suspensions were implanted site of lesion site from 2–11 create a hemi-section of 2 mm. human that sowed into gel foam and weeks only. between 18 2.5–5  105 MSCs suspended in Axonal growth at the site of and 45 years gel foam were directly and injury was MSCs’ donor Recipient: immediately injected post SCI. dependant. SpragueDawley rats Transplanted HSCs integrated Lumbar (L1–L3 level): Excision Donor: Human 2  105 CD34 + HSCs were of spinal neural tube segment Recipient: inoculated directly into the into the spinal cord. was made by stretching. injury post SCI. NeuN and MAP was expressed

Reference

Chopp et al. [26]

Sasaki et al. [27]

Hofstetter et al. [28]

Akiyama et al. [29]

Wu et al. [30]

Ankeny et al. [31]

Ohta et al. [32]

Substantial progress on Zurita and motor functions post 15 days Vaquero [33] of transplantation, which until three months before transplantation did not show any functional recovery. No significant functional Lu et al. [34] recovery was seen in the injury sites of recipients.

Significant improvement on BBB scores of the recipients was observed.

Neuhuber et al. [35]

Transplanted MSCs revealed suggestive neuronal active

Sigurjonsson et al. [36]

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Table 1 (Continued) S. N.

12

13

14

15

16

17

18

19

Injury or Lesion

Donor/ Recipient species

Embryonic chick Thoracic (T10 level): Lesion was Donor: Wistar created using balloon-induced rats Recipient: Rat compression, where a small hole was made by inserting a catheter into the epidural space, wherein a balloon was expanded for 5 min to create paraplegia. Donor: Human Thoracic (T8–T9 level): modified balloon compression Recipient: technique was used, where the Male Wistar paravertebral muscle over L1– rats T8 level was detached for removal of soft tissue and corresponding process of spines. Catheter was then inserted as above to create lesion. Donor: Human Thoracic (T9–T10 level): Laminectomy with rod Recipient: Female weighing 10 g was dropped Spraguefrom 12.5 mm height (mild Dawley rats lesion), 25 mm height (moderate lesion) and 50 mm (severe lesion).

Transplant

Histological signals

Functional consequences

Cultured cells (Rat BMSCs) were given intravenously after a week of lesion.

by HSCs, including axonal and dendritic structures. Inoculated MSCs migrated towards injury, leading a hypotensive MRI signal. Cavity formation was found to be reduced on the lesion sites.

membranes and synaptic potentials. Substantial development on Sykova’ and locomotory scores of grafted Jendlova’ [37] animals compared to control.

Substantial improvement on 1,000,000 cultured MSCs were Transplanted MSCs had introduced slowly on the right entered the ventrolateral site locomotory scores, after femoral vein after 7 days of SCI. mostly and also towards injury transplantation. epicentre. Some of these differentiated to oligodendrocytes, but not into NeuN. Axonal remyelination was seen post transplantation. 5  105 cells were inoculated directly into mild and severe lesions after 7 days of SCI 1 106 were directly injected into the moderate lesions post a week of SCI.

Thoracic (T6–T8 level): Laminectomy using 25 g weighing cylinder of 12-mm2 diameter was released from a 20 cm height creating a necrotic centromedullary lesion. Cervical (C4–C5 level): hemisection subtotal of spinal cord in all rats.

Donor: Male Wistar rats Recipient: Female Wistar rats

Post 3 months of SCI, 3  105 cells were inoculated into the injury either by intravenous route or directly.

Donor: Healthy Human Recipient: Female SpragueDawley Rats

1 106 cells were given by puncturing lumbar and by intravenous method. For direct transplantation, 4.5  105 MSCs were inoculated in the lesion as soon as lesion was made.

Cervical (C3–C4 level): cervical dorsolateral funiculotomy was performed for exposing one of the spinal cord segment by partial laminectomy where dorsal columns and ventral funiculus where kept intact. Thoracic lumbar (T13–L2 level): silicone balloon catheter compression method was used to create a lesion, wherein a catheter was inserted and balloon was then inflated in extradural space of spine.

Donor: Human 1.5  105 cultivated cells were Recipient: Rat given parenterally into the injury after SCI immediately.

Thoracic (T8–T9 level): Laminectomy and balloon compression technique was used for creating lesion.

Donor: Adult female Wistar rats Recipient: Adult female Wistar rats

Donor: Beagle Dog Recipient: Beagle Dog

Reduced cavity formation was observed in both mild and moderate cases, as the sites were filled with cellular materials. Also, increased Schwann cell and oligodendrocyte migration was observed in moderate lesions. IV route transplantation showed MSCs which formed bundles around the injury that possess neurofilament and decreased the cavity size in all the rats. Amongst the three ways, lumbar puncture delivery of cells showed better engraftment of cell compared to others. Cavity had reduced in size and tissue sparing had increased in all groups. Cavity had reduced in size and tissue sparing had increased.

In both the allogenic and autologous group, mRNA expression for neurotrophic factors after MSC transplantation was increased compared to Control group. Post 5 weeks of injury, size of cavity had decreased considerably in both the case of autologous and allogenic in comparison of control. Three groups of rats, containing Volume of injured area of seven rats in each group, NIMSCs treated groups was randomly selected was used for significantly less in comparison the protocol. to Olfactory ensheathing glial In group I, 1 106 NIMSCs and cells (OEC) and control groups. in group II, 1 106 OECs were inoculated into the lesion site at 3 points (5 ml at each site of injection). In group III, which served as control, 1.5 ml of medium without cells was inoculated. Thirty dogs in total, consisting 10 dogs in each group, i.e. control, autologous and allogenic group were used for study. Density of 1 107 prelabelled cells were injected into the spinal canal via lumbar puncture after a week of SCI.

Reference

C’ı9zkova’ et al. [38]

Substantial development was Himes et al. seen on locomotory scores of [39] the recipients. Tentative nurturing and sensitivity to heat was found in the moderately injured cases.

Substantial progress on locomotory scores, and this was much better in direct transplantation compared to intravenous.

Vaquero et al. [40]

Functional assessments were not noticed.

Paul et al. [41]

Functional assessments were not seen.

Samdani et al. [42]

Autologous MSCs was found to be more therapeutically beneficial in comparison to allogenic MSCs. Also, autologous group had more olby scoring than allogenic group.

Jung DI et al. [43]

NIMSC is more effective and showed significant improvement on BBB scores, when compared to OEC and control groups.

Yazdani et al. [44]

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Table 1 (Continued) S. N.

Injury or Lesion

Donor/ Recipient species

Transplant

20

Dorsal laminectomies (T9 level): SCI device of 2N weight and 2.5 mm tip was dropped to create lesion.

1.5  104 cultured MSCs were directly inoculated post SCI immediately into the injury.

21

Laminectomy was done at T8– T9 level.

Donor: Young adult GFP transgenic rats Recipient: Male SpragueDawley rats Donor: Male Sprague Dawley rats Recipient: Female Wistar rats Recipients being divided into five groups (I–V)

22.

Laminectomy was done at the thoracic level of T12, by using drop weight method.

Donor: Male Wistar rats Recipient: Male Wistar rats

23.

Laminectomy was done at T9– T10 level using drop weight method, to create moderate to severe SCI.

Donor: Male Albino rats Recipient: Male Albino rats

24.

Lesion of size 3.0 mm  4.0 mm was created at T12 level, using modified Allen’s impact method.

Donor: Wistar rats Recipient: Female Wistar rats

25.

Laminectomy at the T7–T8 level Donor: of the rats, using weight drop Female Spraguemethod. Dawley rats Recipient: Adult male Sprague Dawley rats Laminectomy was made at T8– Donor: Healthy T10 levels, using incision human method. Recipient: Male Sprague Dawley rats

26.

Histological signals

Recipients showed reduced blood-spinal cord barrier leakage, increased von Willebrand Factor and endothelial barrier antigen expression. Myeloperoxidase enzyme was 3  105 cultured BMSCs was given intraspinally. significantly decreased along Group I served as control with the elevated expression of group, where injury was made VEGF, BDNF was seen in the but not treated. group V, when compared to Group II served as SCI group, others. Also, Group III and Group IV where only serum was given. Group III served as BMSCs showed good results when group, where only cell compared to other two groups I transplantation was done. and II. Group IV served as minocycline group, where only drug minocycline was given at a dose of 50 mg/kg. Group V: BMSCs + Minocycline group, where both BMSCs and minocycline was given. 1 106 BMSC was injected via BBB scores was assessed for the tail vein of injured rats one one month period in the hour post SCI. injured rats. Rats were divided into five After SCI, rats showed severe groups as above, but the paraplegia. medicine used was dantrolene.

Functional consequences

Reference

MSC treated rats showed improved locomotory functions after two weeks of treatment.

Matshushita et al. [45]

Chen et al. Group V showed significant increase in the BBB scores [46] when compared to other groups, although Group III and Group IV had also showed increase effects. Even the lesion had improved in these groups.

BBB score had improved significantly after the third week of treatment. Neurological improvements were seen in BMSC treated group, BMSC + DAN treated group and only DAN treated group. The effects were almost similar in all these groups. 107 BMSCs was transplanted Locomotor functions had Histological examinations into the injured rats either showed expression of more glia improved significantly post 5 intravenously or intralesion. cells and lessened dark weeks of SCI treatment with neurons, mild vacuolation in transplants of BMSC. intravenously treated groups, while in intra lesion treated group, neurons were visible clearly but no vacuolation was observed. Also, TGF-ß gene was significantly decreased in treated groups. Strong myelin sheath was formed in the spinal cord of intralesion treated group. 4  105 MSC was given to the Motor functions assessed by Migration of the BMSC to the each injured rats via tail vein. injured site was observed in BBB scores showed significant treated rats, along with improvements in the BMSC significant recovery in the treated groups, when treated groups pathologically, compared with controls. when compared to controls. 1 106 labelled BMSCs was BBB scores was significantly MRI scanning showed the injected to the rats after a week migration of transplanted cells higher in the treated groups of SCI lesion. in the injury site. than the untreated ones, post BMSC transplants showed 3 weeks of SCI. improvement in the axonal recovery, when compared to controls. 1.5  105 cells of human clonal MSCs were injected into the injured epicentre.

forms fibrotic scars. Once such fibrotic scars are formed, it block leukocytes and macrophages invasion, directly into the injury

Cavity was almost filled when compared to the control group. Axonal regeneration was increased in the cell treated groups, also secondary pathogenesis was reduced in treated groups, when compared to controls.

Junta Torres et al. [47]

Elawady et al. [48]

Jia et al. [49]

Zhang et al. [50]

Kim et al. [51] BBB scores had improved slightly in the treated groups but there was no significant differences, when compared to controls.

during early stage of injury and hence, is responsible for the glial scars [66–68]. Thus, any SCI model is healed when fibrotic scar

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Table 2 Clinical response on SCI patients after mesenchymal stem cells transplants derived from bone marrow and the responses observed during follow-up. S. N.

Patient group

1

Six patients with Grade A complete cervical SCI. Acute patients were transplanted with 1.98  109 autologous MCPs was directly inoculated into the injury after a week of SCI. After performing laminectomy for transplantation to the injured site, five cycles in total (first 5 days of months, over a period of 5 months in total) of Granulocyte-macrophage colony stimulating factor (GM-CSF) was inoculated subcutaneously. No control group was included in the study.

Park et al. [52]

2

Amongst ten patients chosen for study, 7 had paraplegia and 3 had quadriplegia. Mean injury period of patients was 3 years.

Callera and Naseimento [53]

3

Twenty patients in total, amongst which 15 had complete SCI of Grade A and 5 had incomplete SCI (4 ASIA B and 1 ASIA C) were used for this protocol. Twelve cervical and eight thoracic cases were in total.

4

Randomly selected 48 patients who had complete SCI grade A were enrolled for the study. Amongst these, 30 were cervical case and 18 were thoracic.

5

Nine patients with grade A SCI of more than 6 month post injury were taken for the study. Amongst nine, 6 were cervical and 3 were thoracic case.

6

One patient of ASIA A SCI was taken for this protocol.

7

Total eight patients, where five case had SCI ASIA A grade SCI while three case with incomplete SCI (1 ASIA B grade SCI and 2 ASIA C grade SCI) were selected for this study. All the cases were thoracic.

8

A total of 30 persons with Grade A cervical injury and thoracic injury were taken for the study. Group 1 include victims with less than 6 months post injury. Group 2 include victims with more than 6 months post injury.

Transplant

Follow-up

Follow-up duration was from 6 months to 18 months. Neurological functions had improved on five amongst six patients (one improved from Grade A to B and four from A to C) Although GM-CSF administration induced fever, myalgia pain, and leucocytosis. Any serious complications, such as, mortality and morbidity were not found Neurological functions were not aggravated as well. Chronic sufferer (average 3 years after SCI) were Sensory and motor functions after inoculated with 1.98  109 autologous MCPs via transplantation did not show any significant lumbar puncture into the cerebrospinal fluid improvements. No adverse events were reported. (CSF). No control group was enrolled for the study. Out of twenty patients, 7 acute and subacute Follow-up period was kept at 3, 6 and 12 months patients (10 days to 1 month after SCI) and 13 post transplantation. chronic patients (2 months to 17 months after Amongst 6 patients, who received intra-arterial SCI) were transplanted with 104  55.3  108 transplantation, all 4 subacute patients and only autologous MCPs injection via arterial route (n- 1 out of 2 chronic patients revealed significant 6) or via veins (n-14). improvement on neurological function. There were no control group enrolled. Of the remaining 14 patients who received transplantations via i.v. route, only one patient had improved on electrophysiology and ASIA score. No adverse events were reported. Patients were enrolled in three groups such as; Post-surgery, average duration of follow-up was acute (n = 17, SCI of <14 days), subacute (n = 6, 10 months. SCI of 14 days to 8 weeks) and chronic (n = 12, In total, acute (29.5%), 33.3% of subacute (33.3%), SCI of >8 weeks) who were given 2  108 none from chronic (nil) and control (7.7%) patients had significant improvement on autologous MCPs inoculated directly into the injury. neurological functions. After surgery, five cycles in total (first 5 days of Granulocyte macrophage colony stimulating months, over a period of 5 months in total) of factor administration induced fever, facial Granulocyte-macrophage colony stimulating rashes/flushing, and headaches but there were factor (GM-CSF) was inoculated no adverse events. subcutaneously. Some patients in both the treatment and control Control group included 13 patients who were groups also experienced neuropathic pain. treated with conventional decompression and fusion surgery. Follow-up period was 365 days. Each patient was given between 20 and 67 million autologous cells in a gel foam at multiple Post-transplant, victims showed improvements locations into the injury directly, in a carrier gel on neurological function (1 from ASIA A to B and foam covering the injury. 8 from ASIA A to C). There was no any inclusion of control group. No any adverse events were reported. Follow-up duration was till half a year. Victim was transplanted with 3.1 107 cultivated cells via lumbar puncture. Neurological functions had significantly No control was included. improved post one and third months of transplantation. However, between 3 and 6 months, only slight improvement on motor functions was observed. No any adverse events had occurred. Follow-up period was two years. Four acute patients (5 days–7 months post injury) and four chronic patients (5 years–21 Amongst 4 acute patients, 3 showed significant years post injury) were inoculated with an improvement on neurological functions grade A average of 4  108 cultured cells. to Grade C while amongst 4 chronic patients, Cells were given via lumbar puncture and only 3 showed improvements (one from Grade intravenous route at various positions of the A to C, one from grade B to C and one from grade cavity after removal of glial tissue and the glial C to D). scar tissue removal and freeing of spinal cord. All the patients showed increased scores on There was no control group enrolled in this quality of life and bladder sensation plus its study. control. There was no any report of serious complications. 6 1 10 cultured autologous cells, per kg of body Follow-up time was 1–3 years of weight of patients, were given by lumbar transplantation. Only 10 patients followed during first year of puncturing. There were no any control group taken. treatment, 10 others during second year and three others during third year post treatment. Group 1 patient showed some improvement on neurological scores when compared to Group 2 patients. No cases of any adverse events were known.

Reference

Sykova et al. [54]

Yoon et al. [55]

Deda et al. [56]

Saito et al. [57]

Geffner et al. [58]

Pal et al. [59]

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Table 2 (Continued) S. N.

Patient group

Transplant

Follow-up

9

Sixty-four patients were taken with an average of 3.6 years post SCI, both complete and incomplete cases of SCI were taken for the study.

Minimal neurological functions were found to Kishk et al. be improved in the treated groups on [60] comparison with control. No substantial changes had occurred clinically, in between two groups.

10

Thirteen patients with chronic complete SCI of >8 weeks duration were taken for this protocol.

11

Six patients, one with cervical lesion (C5-C6) and five patients with thoracic lesion and ASIA Grade A (n = 3), B(n = 2) and C (n = 1), were taken for this protocol.

Amongst 64, 44 patients received autologous BM-MSCs administration, for six months, via intrathecal route. All subjects were given a rehabilitation therapy, thrice in a week. Twenty patients did not agree for the treatment and thus, were treated as control. Dose between 3 and 8 million cells/kg body wt. was given. 1st dose was between 1 and 4 million cells/kg body wt. that was inoculated directly at the site of lesion. 2nd and 3rd dose was between 1 and 2 million cells/kg body weight and was given via lumbar puncture. Surgery was done to open dura and to expose the spinal cord, under general anaesthesia. Autologous activated Schwann cells (AASCs) at the concentration of 2–3  104 was injected into the injured area directly, and the dura was closed with sutures after injection. The grafting of cells was done ranging from 1 week of injury to 20 months of injury, depending on different patient conditions.

12

70 patients, with chronic cervical and thoracic SCI of not less than 12 months were selected for the study. The study was single blind clinical trial.

13

A 15- year girl patient with complete SCI at T2-T3 level was chosen for the study of safety and efficacy of MSC transplantation.

14

18 patients, out of which 13 were male and 5 were female, with complete motor deficits and paraplegia, at the lumbar and thoracic region, were included in the study, from March 2012 to December 2014. Patients were devoid of any muscle atrophy and psychiatric problems. 12 patients had thoracic injury while 6 had lumbar injury.

Reference

Bhanot et al. Follow-up period was 1 year. Amongst 13 patients, 1 showed slight [61] improvement on motor function, 2 showed slight improvement in pin prick sensation beneath injury site and 1 showed improvement on bladder fullness sensation.

Three days after operation, cell identification, functional improvement and MRI variation was studied in these six different patients. AASCs exhibited antigen expression. Motor and sensory function was improved after 3–10 weeks post transplantation. ASIA grade improved from Grade A to B in three patients, Grade B to C in two patients and Grade C to D in one patient. Also, the sensory scores had improved in all the patients. MRI scan showed repairment in the cord. No adverse events like tissue loss, glial scar and cyst formation were seen. ASIA impairment scale, electrophysiological 2  105 cells/kg autologous BMSCs was given intrathecally via lumbar puncture. somatosensory potential, MRI and motor and Physical therapy along with autologous BMSCs sensory functions were evaluated for was given to 50 patients, while only physical comparison during study. therapy was given to remaining 20 patients Patients treated with physical therapy alone which served as control, and comparison showed less improvements than the ones with between two was made. BMSCs + physical therapy group. No adverse events were reported in the cell treated patients. Thoracic patients showed better improvements than the cervical patients. ASIA grade had improved in cell treated patients as well. 1.54  108 autologous BMSCs was given during During 2 years of treatments, no any adverse the treatment in total, divided as: events were seen or reported. 3.2  109 via parenterally, ASIA score improved from A grade to D grade. 9 0.5  10 via intrathecally, both given post 10 Motor and sensory functions were restored, weeks of SCI, and along with MRI revealed the recovery of injury 1.3-3.65  107 via lumbar puncture, given every at the thoracic level. 3 to 4 months post SCI. 750 million bone marrow cells, suspended in Follow up period was 12 months post 2 ml of saline, was given intrathecally. transplantation. Evaluation was made based on ASIA impairment scale and by assessing electrophysiological parameters. The motor and sensory functions had improved in 50% patients, wherein seven patients had improved ASIA grade by 1 grade while two patients had improved by 2 grade. Five out of twelve patients with urinary tract infection and seven out of nine patients with intestinal problems showed improvement. No any adverse events were reported in any case.

Zhou et al. [62]

El-kheir et al. [63]

Jarocha et al. [64]

Zurab et al. [65]

Note: ASIA stands for American Spinal Injury Association Impairment Scale, where ASIA class or grade A means complete lack of motor and sensory function below the level of injury (including the anal area), ASIA B means some sensation below the level of injury (including the anal sensation), ASIA C means 50% of the muscles that lies below the level of injury cannot move against gravity, ASIA D means more than 50% of the muscles lying below the level of injury can move against the gravity and ASIA E means all the neurologic function has returned back to normal.

formation is reduced along with reduced glial scar due to the regeneration of axons. And BMSC is responsible mostly in reducing fibrotic scar and glial scar, thereby treating both the complete acute SCI and even in chronic SCI of animal models.

When BMSC transplantation in human is considered, it was observed during clinical trials that BMSC given subcutaneously was effective for acute complete case of SCI in humans [52]. Also, lumbar puncturing could be a way of delivery method but the

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effect was not found to be significantly higher [53]. Similarly, the intra-arterial method of cell transplantation provided more therapeutic benefit in the ASIA improvement scale, when compared to the intravenous method [54]. Also, when the case of SCI is severe and cell loss is magnificent, then only BMSC cannot be therapeutically effective, and in such case, the gap left for bridging has to be replaced with more number of cells so as to fill the extracellular matrix in the site of lesion. Thus, treating complete chronic case of SCI is doubtful with only transplantation. It has been reported than even complete SCI in patients can be treated with transplantation of BMSC [55]. Also, the ASIA score had improved from A to B/C grade when autologous BMSC is given either intravenously or directly to the lesion [56]. When an intrathecal route of administration was used in patients, the ASIA grade had repaired to grade B/C/D during a year follow up [57]. In patients with acute SCI and even chronic SCI, routes such as intraarterial and intravenous administration of mononuclear cells in the acute SCI patients, has been proved to be safe, effective and reliable as well [58]. Also, the intravenous and repetitive intrathecal routes of transplantation is an effective route for treating SCI cases [64]. More detailed examinations is required to evaluate the effect and the best route for treating SCI. The use of intrathecal route for the transplantation of BMSC into the SCI case of less than six months was proved to be beneficial than SCI post six months [59]. The use of autologous BMSC in acute, sub-acute (any delivery method) and chronic case (direct injection) of SCI showed positive effects except with the chronic SCI [61]. The autologous BMSC is a safe and feasible technique [65] in most of the cases but this should be contraindicated in patient with myelitis [60]. This could be an arena for further research to the interested researchers. Also, the use of BMSC in chronic traumatic SCI needs further study in randomized controlled trails and preclinical models. Local and repetitive transplantation of cells in the lesion has been proved to be more effective than the systemic transplantation [63]. When compared with cell transplantation in acute SCI, delayed cell transplantation either in chronic SCI patients or perhaps in the subacute setting is likely to be more successful due to a more permissive injury environment when the inflammatory response has subsided, but the effect is not as high as in acute and sub-acute condition. Thus, in future more research has to be carried out on the clinical trial efficacy of SCI in patients, with acute, sub-acute and chronic case of SCI and mostly with the use of autologous BMSCs. 5. Conclusion Various animal studies performed and various clinical trials performed in humans, with the use of bone marrow stem cells transplants, as shown in Tables 1 and 2, suggest that the mesenchymal cell therapy can be a promising candidate for patients, with acute, sub-acute and chronic conditions of injury. Mesenchymal cells derived from bone marrow have good healing potential for treating various levels of the SCI. However, more research and clinical trials with double-blind, random controlled and more number of subjects are required for further justification of the effectiveness of the therapy on treating spinal cord injury of various origins. Various studies have been suggesting that the transplanted BMSCs can differentiate into a myelin-forming cells and thus repair demyelinated spinal cord. Also, it is well established that as per the local host environment that surrounds injury, cell gets differentiated into that particular cell type, thereby repairing the injury. When transplanted in vivo, BMSCs that was devoid of debris, erythrocytes and platelets repaired the created lesion [27]. Not only this, when the transplantation was done after some period of

gap rather than immediate transplantation post-SCI, the results was found to be more effective on the former case. This can be due to the later phase of SCI which comes after a gap of sometime post injury, being more hostile to the transplanted cells as compared to early phase, when presence of toxic compounds, lytic enzymes and free radicals at the surrounding environment suggesting defence mechanisms to any foreign cells [28]. Cell therapies have wide therapeutic applications in treating the SCI. MSCs obtained from bone marrow hold a major therapeutic potential due to the easiness for its separation from even a small number of bone marrow extracts along with its significant anti-proliferative, anti-apoptotic and anti-inflammatory features. The precise way of efficacy of stem cells in functional recovery is not yet clear. However, BMSCs are supposed to be immunosuppressive and there are increasing evidences for the same as well. These immunosuppressive properties may be responsible in decreasing the lesion cavity as well as in decreasing microglia/macrophage reactions. Not only that, BMSCs transplantation at the site of SCI may enhance tissue preservation as well as it can reduce sensitivity that is induced by injury on triggering of mechanical stimuli in experimental SCI model, suggesting antiinflammatory property of BMSCs [69–72]. However, in future more research works have to be performed to justify for the same in the immune system. BMSCs that are used in SCI are also shown to recover CNS functions and this is supposed to occur by openly transforming the SCI environment directly, wherein regeneration of axons is seen (reduced glial scar in an indicative of this). Various neurotrophic cytokines are synthesized on BMSCs transplantation, which supports and stimulate growth of neurons, including vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) [73,74]. However, these neurotrophic factors have limited effect on the milieu of spinal cord. In one study, BMSCs when administered to the injury of spinal cord, it increased the production of neurotrophic factors like cytokines, including interleukin-6, interleukin-7 and VEGF. Each of these factors play a vital role in repairing the injury as well as in repairing the secondary tissue [75]. Thus, the interactions between transplanted MSCs and SCI environment, is very complex to elucidate and also can be a complex area for future scientific research. If the effect of cell delivery method has to be concerned, then gene modified MSCs serves better purpose of repairing SCI compared to only MSCs. This might be due to cytokines produced by MSCs, some of which are, insulin like growth factor (IGF), BDNF, VEGF, GM-CSF, fibroblast growth factor and TGF, which have an immunomodulatory effect, and thus extremely promising candidate for repairing multiple injuries related to neurons, but the levels of these cytokines are not as effective as that with the genetically modified MSCs or tissue engineering. When MSCs are genetically modified, it increase the proteins secretions, which enhance the repairment of injury of spinal cord and also promote their survival and regenerating potential of neurons. Various proteins involved with repairment of SCI involves: neurotropic factors such as neurotropin 3, BDNF, GDNF, NGF, kinase C and hepatocyte growth factor [76]. Research related with genetic modifications of MSCs in animal models of SCI, suggested that there was significant improvement on locomotor and electrophysiological functions, when transduced genes contained neurotropin 3 protein by using adenovirus as a gene carrier’s vector [77,78]. Not only these, various gene carriers used in many researches, reports of using viral vectors like retroviral, adenoviral, and lentiviral vectors. However, non-viral vectors has more advantages of being less toxic, immunogenic and with unlimited transgene size. Thus, non-viral vectors can be more beneficial for SCI repair by transducing exogenous genes into BMSCs [79]. But the limitation of using non-viral vectors is that DNA or RNA has to be protected

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from degradation until efficient delivery of genes into the target cells [80]. Thus, more research with modified MSCs using non-viral vectors have to be performed to elaborate on the underlying mechanism and explanation of its success. This can be a new area of research for further academics and institutions who are interested in SCI repairment area. In one study, it is suggested that the ideal patients who can get the most benefit out of MSCs therapy are the one with injury at cervical and thoracic level with an incomplete clinical syndrome, i.e. with the evidence of residual electrophysiological function despite of complete SCI and also without any glial scars or compression of spinal cord, but unfortunately, all these conditions ideally do not meet the SCI patients’ conditions [81]. Thus, what we can conclude is both acute and chronic SCI can be treated with MSCs, but mostly the ASIA A and B are treatable with BMSCs, as shown by the references in the tables above. Thus, the BMSCs can be a promising therapy in the repairment of SCI provided more data related to phase 1 and phase 2 trials are available and more research is focused in these area. Thus, MSCs can provide a hope for treating SCI both to the patients, and the families belonging to such patients. Financial and competing interests’ disclosure The authors have no financial interests or affiliations in any organizations and there are no conflicts of interests resulting in this manuscript. During the making of this manuscript, no writing assistance was taken. Conflict of interest The authors report no conflicts of interest in this review. This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] R.J. Dumont, D.O. Okonkwo, S. Verma, R.J. Hurlbert, P.T. Boulos, D.B. Ellegala, A. S. Dumont, Acute spinal cord injury, part I: pathophysiologic mechanisms, Clin. Neuropharmacol. 24 (2001) 254–264. [2] WHO Factsheets on Spinal Cord Injury, (2013) http://www.who.int/ mediacentre/factsheets/fs384/en, (Accessed on 16.10.16).. [3] L.H. Sekhon, M.G. Fehlings, Epidemiology, demographics, and pathophysiology of acute spinal cord injury, Spine J. 26 (2001) S2–S12. [4] National Spinal Cord Injury Statistical Center, Facts and Figures at a Glance, University of Alabama at Birmingham, Birmingham, AL, 2016. [5] C.K. Geng, H.H. Cao, X. Ying, H.L. Yu, Effect of mesenchymal stem cells transplantation combining with hyperbaric oxygen therapy on rehabilitation of rat spinal cord injury, Asian Pac. J. Trop. Med. 8 (2015) 468–473. [6] T. Yilmaz, Y. Turan, A. Keleş, Review: pathophysiology of the spinal cord injury, J. Clin. Exp. Invest. 5 (2014) 131–136. [7] S.S. Park, Y.J. Lee, S.H. Lee, D. Lee, K. Choi, W.H. Kim, O.K. Kweon, H.J. Han, Functional recovery after spinal cord injury in dogs treated with a combination of matrigel and neural-induced adipose-derived mesenchymal stem cells, Cytotherapy 14 (2012) 584–597. [8] Z. Zhilai, Q. sujun, D. Chao, S. Benchao, H. Shuai, Y. Shun, M. Biling, Z. Hui, Preconditioning in lowered oxygen enhances the therapeutic potential of human umbilical mesenchymal stem cells in a rat model of spinal cord injury, Brain Res. 1642 (2016) 426–435. [9] J. Liu, D. Han, Z. Wang, M. Xue, L. Zhu, H. Yan, X. Zheng, Z. Guo, H. Wang, Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells, Cytotherapy 15 (2013) 185–191. [10] V.R. Dasari, K.K. Veeravalli, D.H. Dinh, Mesenchymal stem cells in the treatment of spinal cord injuries: a review, World J. Stem Cells 6 (2014) 120– 133. [11] C.H. Tator, M.G. Fehlings, Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms, J. Neurosurg. 75 (1991) 15–26. [12] R. Vawda, M.G. Fehlings, Mesenchymal cells in the treatment of spinal cord injury: current & future perspectives, Curr. Stem Cell Res. Ther. 8 (2013) 25–38. [13] M.M. Mortazavi, N. Adeeb, N. Hose, R.S. Tubbs, Review: stem cell therapy for spinal cord injury: cellular options, Austin J. Cerebrovasc. Dis. Stroke 1 (2014) 1–5. [14] M.D. Norenberg, J. Smith, A. Marcillo, The pathology of human spinal cord injury: defining the problems, J. Neurotrauma 21 (2004) 429–440.

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