The known-unknowns in spinal cord injury, with emphasis on cell-based therapies - a review with suggestive arenas for research.

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Review

1.

Introduction

2.

Known-unknowns

3.

Conclusion

4.

Expert opinion

The known-unknowns in spinal cord injury, with emphasis on cell-based therapies -- a review with suggestive arenas for research Vidyasagar Devaprasad Dedeepiya, Justin Benjamin William, Jutty KBC Parthiban, Ranganathan Chidambaram, Madasamy Balamurugan, Satoshi Kuroda, Masaru Iwasaki, Senthilkumar Preethy & Samuel JK Abraham† †

Nichi-In Centre for Regenerative Medicine (NCRM), The Mary-Yoshio Translational Hexagon (MYTH), Chennai, Tamil Nadu, India

Introduction: In spite of extensive research, the progress toward a cure in spinal cord injury (SCI) is still elusive, which holds good for the cell- and stem cell-based therapies. We have critically analyzed seven known gray areas in SCI, indicating the specific arenas for research to improvise the outcome of cell-based therapies in SCI. Areas covered: The seven, specific known gray areas in SCI analyzed are: i) the gap between animal models and human victims; ii) uncertainty about the time, route and dosage of cells applied; iii) source of the most efficacious cells for therapy; iv) inability to address the vascular compromise during SCI; v) lack of non-invasive methodologies to track the transplanted cells; vi) need for scaffolds to retain the cells at the site of injury; and vii) physical and chemical stimuli that might be required for synapses formation yielding functional neurons. Expert opinion: Further research on scaffolds for retaining the transplanted cells at the lesion, chemical and physical stimuli that may help neurons become functional, a meta-analysis of timing of the cell therapy, mode of application and larger clinical studies are essential to improve the outcome. Keywords: cell therapies, gray areas, known-unknowns, spinal cord injury, stem cells Expert Opin. Biol. Ther. (2014) 14(5):617-634

1.

Introduction

Spinal cord injury (SCI) is a devastating disease with an annual global incidence of 15 -- 40 cases per million people [1]. The regional data indicate an incidence of 40 per million in North America, 25 per million in Central Asia, 21 per million in South Asia, 16 per million in Western Europe, 21 per million in Sub-Saharan Africa-East and 29 per million in Sub-Saharan Africa-Central [2]. Another disheartening information is that incidence of SCI is on the rise in developing countries due to increase in motor transport. A notable fact is that SCI, which was more prevalent in younger individuals, has increased in the geriatric patients, nearly fivefolds since 1980 and the mortality rate in this group is also considerably higher [3]. In addition to the pain and trauma involved in SCI, there are other severe adverse consequences such as health care expenditures, loss of employment, societal status, etc. making it a burden to the victim, his or her family and even the society [4]. The damage to the spinal cord affects the axons and disrupts myelination, which interrupts the sensory 10.1517/14712598.2014.889676 © 2014 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 All rights reserved: reproduction in whole or in part not permitted

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V. D. Dedeepiya et al.

Article highlights. .

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This manuscript is a brief review of the present status of treatments available for spinal cord injury with emphasis on cell-based therapies. This review has taken into account the various aspects of the approaches toward a successful recovery of injured spinal cord. The gray areas in arriving at a total solution of regeneration have been analyzed under seven categories of vital importance. The expert opinion covers practical solution-centered and priority-wise thrust areas for future research. We consider this manuscript will not only give researchers newer ideas for research but also motivate statisticians and paramedical staff to work toward a meta-analysis for further exploration and confirmation of our stand points.

This box summarizes key points contained in the article.

and motor neuronal transmission to the brain. Though the patients survive after SCI, they are disabled without any promise of cure [5]. The current treatment strategies include surgery to decompress and stabilize the vertebrae, strategies to prevent and manage the secondary complications followed by rehabilitation. In spite of advances in treatment, the neurologic recovery is still limited and nearly all patients face significant neurologic dysfunction and lifelong disability [1]. Cell therapies are considered as a potential option to address SCI. The postulated mechanisms by which cells or stem cells may help treating SCI such as replacement of the damaged cells, remyelination of the spared axons, restoration of nerve circuits, bridging of lesion cavities, secretion of neurotrophic factors, release of anti-inflammatory substances and neovascularization [1] though gives hope, the reality is rather depressing. The expected results are not being observed in human clinical trials [6]. There are several reasons for this and this brief review brings into focus the major known but fewer understood gray areas in SCI bringing forth several clues, which have to be thoughtfully studied by meticulous research thereby enabling us to gain the best out of the most promising tool of cell-based therapies. 2.

Known-unknowns

Known-unknown I -- gap between animal and human subjects

2.1

The successful translation of stem cell applications for SCI from the bench to the bedside is still a Herculean task. Animal models are routinely employed to study the tissue response to injury and to test potential treatments strategies. The most commonly employed models involve either contusion or compression injury, while transection injuries are employed for detailed studies on regeneration [7]. The different animal models used include rats, mice, cats, pigs and non-human primates. There exists a gap between animal models and 618

human clinical trials in SCI. This gap can be attributed to a variety of reasons. Akhtar et al. proposes three major impediments to this bench to bedside translation: i) the differences in the type of injury between the laboratory-induced SCI in animal models and those that occur in clinical settings; ii) the interpretation of the functional outcome in the laboratory models along with the parameters used for interpretation differing between laboratories; and iii) the interspecies and inter-strain differences in the pathophysiology of the SCI [6]. Though the acute gross appearance of the human and animal cords observed after SCI is quite similar, an in-depth analysis of the clinical histopathology, however, reveals an extensive variation of features in the wound healing process (gliosis, connective tissue deposition, sensory fiber growth, demyelination and cyst formation) [8]. Even between closely related species like the rat and mice there exists an important difference in the SCI, wherein rats develop large fluid-filled cystic cavities at injury site while mice don’t. This difference becomes important when studying transplantation strategies. Even among mice, different strains respond differently. For instance, the genetic deletion of neurite outgrowth inhibitorA (Nogo-A), a myelin-associated inhibitor shows more axonal growth in 129X1/svJ mice than in C57BL/6 mice [9]. Jones et al. [10] outline another potentially important difference between the rodents and the human spinal cord, which is the presence of the significant amount of cerebrospinal fluid (CSF) within the intrathecal (IT) space around the human cord that may cushion the spinal cord protecting it from injury. However, a drawback with this is that the pressure waves within this significant amount of CSF at the time of injury may even contribute to the extension and severity of the primary injury. They further postulate that with this background, large animal models may serve as human equivalents in terms of the dimensions and physiology, to a certain extent [10]. In this regard, miniature pigs deserve a mention as earlier studies [9,10] have shown that they serve as an intermediary between rodent and human SCI in studying CSF, spinal cord and dura interactions during injury. Though cats, pigs and non-human primates serve as more equivalent models to human SCI than rodents, they are not commonly used due to factors like size, cost, availability, housing facilities, medical care and ethics. These issues along with other translational issues scale-up including the differences in quantity of stem cells required for animals and that for clinical studies need to be kept in mind. The article by William et al. [11] about intralesional (IL) application of autologous bone marrow mononuclear cells (BMMNCs) embedded in a hydrogel (thermo-reversible gelation polymer [TGP]) scaffold for SCI in a canine model may perhaps be appropriate to be cited in this context because it was not a laboratory-induced injury, but the canine was injured in a road traffic accident similar to what happens in humans. The canine became ambulatory on the 133rd day after autologous stem cells were applied impregnated in a polymer scaffold and was followed up for 2 years. The limitation in this study is that

Expert Opin. Biol. Ther. (2014) 14(5)


The known-unknowns in spinal cord injury, with emphasis on cell-based therapies

there was no control, as maintaining a control animal exactly recapitulating the injury is not possible and another limiting factor is also that the canine might have functionally recovered by itself without any treatment. Though this study may be cited for a human equivalent, there are still several miles to go to recapitulate the exact pathophysiology of clinical SCI in laboratory conditions. The extent to which secondary changes after injury is recapitulated in the animal models is still a major question because of the variations existing in the nature of the primary injury in terms of impact velocity, amplitude or duration [8] between the laboratory animal models and clinical settings. The differences between the age of the animals used as models of SCI and that of human victims are factors that are to be considered. Mostly, laboratory animals are young and do not have associated comorbid conditions. It has been demonstrated that aging and conditions like diabetes lead to impaired cell proliferation, decreased angiogenic potential and decreased wound healing ability of the damage per se as well as that of the stem cells [12]. An article by Beltrami and colleagues opens our knowledge to the cellular senescence aspects in regenerative medicine. They argue that animal models employ animals that are relatively young unaffected by cellular senescence, while clinical studies are undertaken in individuals who may be relatively older and affected by comorbidities that might affect the cellular senescence [13]. This holds good for SCI too as SCI leads to other comorbidities such as obesity, which gives rise to a series of events such as dysfunctions in adipogenesis that leads to bone marrow (BM) alterations, which might eventually lead to cell senescence and the hematopoietic stem cell (HSC) depletion from the BM niche [13]. It is no surprise that the results obtained from stem cell applications in young animal models of SCI in the laboratory are not being observed in clinical human victims of SCI, where the victim might be aged or may have associated comorbid conditions and probably the age-dependant decrease in CD34+ BM -- HSC quantity might also be a factor to be considered as reported by Dedeepiya et al. [14]. To overcome this issue, in vitro selection of cells based on senescence markers and using drug/smallmolecule-based approaches to enhance the in vivo life span of cells could lead to better outcomes. Another important factor contributing to the difference in outcome observed in animal models and clinical SCI is that cell types like the induced pluripotent stem cells (iPSCs) [15,16] that have been applied successfully in animal models have not been translated to clinical settings yet. Even embryonic stem cells (ESCs), which were applied clinically in a clinical trial, could not be studied for long-term outcome as the trial was abandoned [17]. Since only few types of stem cells have been clinically applied for SCI, the gap between animal subjects and human victims can be further understood only when the other cell types are clinically applied. A literature review in 2009 [18] stated that while there are numerous studies assessing functional outcomes of experimental SCI, studies with motor outcomes dramatically outnumber those with autonomic outcomes.

The locomotion in animals that is quadrupedal [19] and the more complex autonomic dysfunctions occurring in human SCI primarily the cardiovascular, bronchopulmonary, urinary, gastrointestinal, sexual and thermoregulatory occurring in high-level cervical injuries [20], which have not been extensively studied in animal models, may also contribute to the gap in the outcome between animal and clinical studies. As a possible solution, animal models of stress urinary incontinence are available to study the outcome of cell therapy on urinary incontinence [21]. Telemetric monitoring of corpus spongiosum penis can be used for assessment of micturition and erectile events following SCI in animal models [22]. Thus, proper methods of assessment of motor, sensory and autonomic functions are essential for preclinical modeling to reduce the gap between these preclinical models and human studies. Markerless motion tracking to assess motor function [23], sensory neural recording interfaces to monitor bladder and limb-state [24], open-field behavior analysis [25] are some of the technologies available, which can be used for various assessments in animal models. Other variables like the time of intervention, gender of the animals used as models, the level of injury (cervical or lumbar), the time duration for which the animals are followed up, all deserve mention as factors to be explored by which the existing gap between animal models and humans could be overcome. 2.2 Known-unknown II -- source of cells used for therapy

Once SCI occurs, the endogenous reparative mechanisms start acting and there is a proliferation of the adult stem cells and progenitor cells (PCs) within the region surrounding the injury. However, the growth inhibitors present on the oligodendrocyte myelin debris and on the cells that form the scar tissue may act against this reparative mechanism thereby resulting in failure of repair of the damaged spinal cord [26]. Hence, cell-based therapies in which autologous and allogenic cells or stem cells are being isolated and/or cultured in vitro and injected either locally or via other routes to reach the site of SCI and help in repair are employed. The cells used in cell therapy for SCI act through different mechanisms such as differentiation into neural cells consequently supporting anatomical or functional recovery, secreting neurotrophic factors that may act as neuro-protective agents or facilitate axon regeneration [26], decreasing inflammation, etc. Table 1 depicts the major types of cell sources employed for repair after SCI such as the ESCs, fetal stem cells (FSC), umbilical cord blood stem cells, olfactory ensheathing cells (OEC), BMMNCs, BM-derived HSCs, BM-derived mesenchymal stem cells (MSCs), neural stem cells (NSCs) and iPSCs, their advantages and disadvantages (selected clinical and experimental studies have been presented). Though several cell types have been postulated for SCI repair, only a few have been clinically applied. The oligodendrocyte precursor cells (OPCs) and neural precursor cells (NPCs) derived from ESCs have been studied in animal


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Deda et al. [37] Several. Some include Park et al. [136], Karamouzian et al. [38], Dai et al. [137], Jiang et al. [138], Pal et al. [139] two clinical trials ongoing [43,44]

Sigurjonsson et al. [132] Several. Some include Yazdani et al. [133], Karaoz et al. [134], Ohta et al. [135], Shin et al. [66], Lin et al. Iwai et al. [40], Cusimano et al. [41], Bottai et al. [42] Kobayashi et al. [16], Fujimoto et al. [46]

Bone marrow-derived hematopoietic stem cells (BM-HSCs) Bone marrow-derived mesenchymal stem cells (BM-MSCs)

Neural Stem Cells (NSCs)

Induced pluripotent stem cells (iPSCs)

[51]

Several. Some include Sykova´ et al. [36], Sharma et al. [129], Park et al. [130], Yoon et al. [131], Geffner et al. [70]

Several. Some include Yoshihara [127], Samdani et al. [39], Raheja et al. [128]

Bone marrow-derived mononuclear cells (BMMNCs)

Huang et al. [35], Mackay-Sim et al. [125]

Ziegler et al. [120], CollazosCastro et al. [121], Yamamoto M [122], Granger et al. [123], Tharion et al. [124]

Ability to support neurogenesis, reduced risk of hypertrophy of the CNS astrocytes, autologous transplantation possible (no graft rejection and no need for immunosuppression), easy accessibility Option of using autologous stem cells and hence lesser chance of immunorejection, Presence of MSCs and HSC in these unfractioned cell population may offer synergistic results in angiogenesis and matrix rebuilding Option of using autologous stem cells and hence lesser chance of immunorejection High neuronal differentiation potential, option of using autologous stem cells and hence lesser chance of immunorejection. Also has immunomodulation potential Tissue of origin similar and hence differentiation potential to neurons is relatively high Personalized cell therapy possible, differentiation potential similar to ESCs

Ease of accessibility

Plasticity of the cells

Seledtsova et al. [30]

Kang et al. [31]

Ability to differentiate into various cell lineages, ability to proliferate over several passages

Advantages

Geron FIH trial (abandoned in 2011) [17]

Clinical studies

Olfactory ensheathing cells (OEC)

[78]

Park et al. [34], Lee et al. [33], Erdogan et al. [119], Ning et al.

Umbilical cord blood stem cells

Fetal stem cells (FSC)

Bottai et al. [108], Kumagai et al. [109], Lowry et al. [110], Cui et al. [111], Kerr et al. [112], Sharp et al. [113], Erceg et al. [114], Salehi et al. [115] Ruzicka et al. [116], Watanabe et al. [117], Tarasenko et al. [118]

Animal studies

Embryonic stem cells (ESC)

Type of cell source

Immunogenicity, high levels of genomic instability

Isolation and directed differentiation difficult

Purification difficult, long-term in vitro manipulation may lead to genomic instability

Purification is difficult, differentiation potential limited

Plasticity is less, in vitro culture difficult, differentiation potential limited

Differentiation potential is limited when compared with ESCs and FSCs, inadequate cell source particularly in autologous transplants, cell purification difficult [126]

Immunorejection (need for immunosuppression) ethical issues, risk of teratoma, though relatively lesser than ESC Need for HLA-matched donors, immunosuppression

Immunorejection (need for immunosuppression) ethical issues, risk of teratoma

Disadvantages

Table 1. Major cell sources employed in the cell-based applications for spinal cord injury (SCI) from the literature (selected clinical and experimental studies have been presented).





models and have shown to result in successful engraftment after transplantation and aid in recovery of limb locomotor function and sensory functions [27-29], but their clinical application has not yet been validated. The only clinical trial employing OPCs from ESCs started by Geron was stopped in 2011 due to non-treatment-related constraints [17]. Fetal neurogenic tissue transplanted into patients has shown to result in a decrease in neurological deficit and improvement of functional independence in 48.9% of the cases. The results were profound in younger patients and in patients who received the therapy within 2 years following the injury [30]. With human umbilical cord blood (hUCB)-derived stem cells, there have not been large clinical trials yet, but there has been one published clinical case report, which demonstrated regeneration of the spinal cord at the injured site and some of the cauda equina below it in a 37-year-old spinal cord-injured female patient [31]. Studies of hUCB-derived stem cells in rat SCI models have shown the migration of the transplanted cells to the lesion area, reduction in lesion size and significant improvement in locomotor function [32]. A number of studies have proven in animal models that transplantation of hUCBMSCs have also resulted in functional recovery [33,34]. OECs are another cell source, which is highly advocated by many scientists as a potential cell source for therapy in SCI mainly because of the characteristics they share with CNS neurons, and furthermore because autologous transplants are possible. Earlier clinical studies using OECs have established longterm safety and efficacy. A study reported that out of 108 patients, who underwent OEC transplantation for complete chronic SCI and followed up for > 3 years, 14 patients (12.96%) improved to ASIA B from ASIA A and 18 patients (16.67%) to ASIA C from ASIA A. The safety was also established in these 108 patients as there were no adverse reactions like cyst formation, tumor, bleeding or any other pathological changes in or around OEC transplant sites [35]. BM is another valuable source of stem cells for cell therapy applications in SCI. From the BM, unfractioned mononuclear cells [36], HSCs [37] and MSCs [38] have all been applied clinically for SCI. Most of these studies gave realistic outcomes mainly establishing safety rather than efficacy [37,38]. One animal study in rats, which compared BMMNCs transplantation and MSC transplantation, observed that in the initial period, mononuclear cells did not evoke any inflammation but MSCs did evoke inflammation. However, the study concluded that at 21 days, the glial scarring and efficiencies of both the groups were comparable [39]. NSCs derived from the fetal and adult CNS have been earlier applied in several animal studies. They have been shown to graft to the area of the injured spinal cord [40] and decrease the inflammation in the injured area by reducing the proportion of activated macrophages in animal models [41]. In another study on a mouse model of SCI, NSCs injected intravenously were shown to improve recovery of hind limb function and attenuate secondary degeneration. In the same study, it was identified that these NSCs retained their proliferation potential even 1 week after transplantation and could

produce neurospheres after recovery from the lesion site and culture in vitro [42]. There are two ongoing clinical trials using CNS-derived NSCs in SCI [43,44]. iPSCs are another major type of cell source, the interest in which has been recently fuelled up by the awarding of the Nobel Prize to Dr. Shinya Yamanaka in 2012 [45]. Neuroepithelial-like stem cells derived from human iPSCs have shown to differentiate into the various neural lineages in the mouse model of SCI and have shown to promote functional recovery of hind limb function [46]. Still, the issues like immunogenicity associated with iPSCs as reported [47] as well as risk of genetic instability after reprogramming [48] need to be addressed before clinical trials are undertaken with iPSCs. Direct reprogramming of skin cells into functional neurons [49], which have been achieved, may also provide hope for futuristic personalized cell-based therapies for SCI. Table 2 summarizes the outcome of a few selected clinical studies on stem/precursor cell applications in SCI. Genetically modified stem cells like MSCs modified to express multineurotrophins [50] or neuroglobin [51] have been demonstrated to increase the functional improvement in animal models of SCI and this option of gene-modified stem cells could also be harnessed in the future for improving the efficacy of cell transplantation in SCI. The possible risks with use of ESCs and iPSCs include potential for tumorigenicity, and immunogenicity of transplanted cells [52,53]. Also, it is important to ensure that patient-derived iPSCs should not retain epigenetic memory of the parent cell [54]. Though differentiated cells derived from pluripotent stem cells have been shown to possess lesser risk for inducing teratogenicity [54], oligodendrocytes differentiated from ESCs have also been shown to possess teratogenicity potential in in vitro studies [55]. Other than potential risks associated, the ethical issues surrounding the use of ESCs due to the issues of destroying the embryo [56] and FSCs particularly from fetal tissues obtained from induced abortions [57], need to be kept in mind while choosing the right cell source. Thus, while studies employing different cell sources offer potential hope for treating SCI, the researchers and patients will be perplexed to choose the precise cell source considering factors like efficacy, differentiation potential, ethical, moral issues, availability and accessibility. Unless a large randomized clinical trial comparing these cell sources is undertaken and issues of ethics, potential risks are appropriately addressed, we will be left with more questions than answers on the true choice of cell source for SCI. Known-unknown III -- time of application of stem cells, route and their dosage

2.3

When time of application of the stem cells is considered, application immediately after injury is marred by the inflammatory reaction occurring in the acute phase, while in the chronic phase, glial scarring is of concern. The glial scaring is due to the macrophages and neutrophils invading the injury site following which apoptosis of oligodendrocytes and Wallerian degeneration occurs. This in turn activates the microglia and astrocytes that results in glial scarring. This


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108 patients with complete chronic SCI

40 patients with complete and chronic cervical SCI; randomly assigned into treatment and control group

Huang et al. [35]

Dai et al. [137]

Autologous BM-MSCs

OEC

Autologous BM-MSCs

Autologous HSCs

Autologous BMMNCs

Cell source

IL

Parenchymal transplantation

LP

IT

IT and IM

Mode of delivery of cells

‘Restoration of the initially absent short-latency SEP in three patients; N20P23 interpeak amplitude increase in SEP elicited by median nerve stimulation (four patients); P38 latency reduction in SEP elicited by tibial nerve stimulation (two patients); appearance of motor-evoked potentials (three patients)’ No adverse effects in any patient. Five patients in the study group and three patients in control group showed recovery but results were statistically borderline Averaged motor scores increased from 37.79 ± 18.45 to 41.25 ± 18.18 (p < 0.01), light touch scores from 50.32 ± 24.71 to 55.90 ± 24.46 (p < 0.01), pin prick scores from 50.53 ± 24.92 to 54.53 ± 24.62 (p < 0.01)’. 14 out of patients became ASIA B from ASIA A; 18 out of 108 patients became ASIA C from ASIA A. Nine patients had improvement in walking ability; 12 of 84 men had improvement in sex function In the treatment group,’10 patients had a significant clinical improvement in terms of motor, light touch, pin prick sensory and residual urine volume, while 9 patients showed changes in AIS grade’

100% of SCI cases showed improvement with respect to muscle strength, urine control, spasticity

Outcome

BM: Bone marrow; HSC: Hematopoietic stem cells; IL: Intralesional; IM: Intramuscular; IT: Intrathecal; IV: Intravenous; LP: Lumbar puncture; MNC: Mononuclear cells; MRI: Magnetic resonance imaging; MSC: Mesenchymal stem cells; OEC: Olfactory ensheathing cells; PBSC: Peripheral blood stem cells; SCI: Spinal cord injury; SEP: Somatosensory-evoked potentials.

11 patients having complete SCI and 20 patients as control

71 children suffering from neurological disorders such as muscular dystrophy, cerebral palsy, and injury to the brain and spine 20 adult patients with chronic SCI at C4--C8 level

Patient characteristics

Karamouzian et al. [38]

Frolov et al. [140]

Sharma et al. [129]

Publication reference

Table 2. Findings of few selected clinical studies on application of stem cells for SCI from the literature.




6 patients with thoracic SCI (T4 -- T10), ASIA A classification, 3 in study group; 3 as control

35 complete SCI patients, American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade A included in study group -- 17 acute, 6 subacute and 12 chronic SCI. 14 patients as control

1 patient with compression fracture of the T12 vertebra and fracture dislocation of the T11, T12 vertebrae

4 acute and 4 chronic SCI patients

Mackay-Sim et al. [125]

Yoon et al. [131]

Kang et al. [31]

Geffner et al. [70]

Autologous BMMNCs

UCB-derived MSCs

Autologous BMMNCs after GM-CSF stimulation

Autologous OECs

Autologous PBSCs

Cell source

Multiple routes: IL, IT and IV

1 million cells injected into sub-arachanoid space of normal spinal cord and another 1 million in intradural and extradural space of the injured spinal cord

IL

IL

Infusion through arteriography

Mode of delivery of cells

26 patients showed recovery of SEP to peripheral stimuli after 2.5 years of follow-up No adverse effects in any patient. No significant functional changes in any patients. In one patient there was an improvement over three segments in light touch and pin prick sensitivity bilaterally, anteriorly and posteriorly AIS grade increased in 30.4% of the acute and subacute-treated patients (AIS A to B or C), but no significant improvement observed in the chronic SCI group. At 4 months, the MRI analysis showed the enlargement of spinal cords and the small enhancement of the cell implantation sites, but they were confirmed to be not any adverse lesions such as malignant transformation, hemorrhage, new cysts or infections Improved sensory perception and movement in the patient’s hips and thighs within 41 days of cell transplantation. CT and MRI results showed regeneration of the spinal cord at the injured site and some of the cauda equina below, posttransplantation Improvements in ASIA, Barthel, Frankel and Ashworth scoring observed

Outcome

BM: Bone marrow; HSC: Hematopoietic stem cells; IL: Intralesional; IM: Intramuscular; IT: Intrathecal; IV: Intravenous; LP: Lumbar puncture; MNC: Mononuclear cells; MRI: Magnetic resonance imaging; MSC: Mesenchymal stem cells; OEC: Olfactory ensheathing cells; PBSC: Peripheral blood stem cells; SCI: Spinal cord injury; SEP: Somatosensory-evoked potentials.

39 patients with complete cervical and thoracic SCI

Patient characteristics

Cristante et al. [141]

Publication reference

Table 2. Findings of few selected clinical studies on application of stem cells for SCI from the literature (continued).



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astroglial scar, whether it surrounds a cyst or not, has been long recognized to be a barrier to axonal growth and regenerative processes [58]. Nishimura et al. [59] compared the injection of neural stem/PCs (NS/PCs) in rats in the subacute and chronic phase of injury. Their results showed that there was no difference in the survival rate of the grafted cells between the subacute and chronic SCI groups. The expression of genes encoding inflammatory molecules was also the same. However, the distributions of the transplanted cells were concentrated around the lesion in the chronic group due to the extensive glial scarring but were more widely distributed in the subacute SCI group. Further, functional recovery was markedly higher when the NS/PCs were transplanted in the subacute phase than in the animals in the chronic phase of SCI. This study throws light on the fact that improved outcomes seen in subacute phase of SCI in earlier studies is mainly due to the increased migration of cells and lesser glial scarring [59]. Also, when the cells are transplanted in the subacute phase of injury, they help in increasing the number of spared/regenerated supraspinal fibers, reducing cavitation and enhancing tissue integrity. These mechanisms could also contribute to the improved outcome in cell transplantation during subacute SCI [60,61]. In chronic SCI, there occurs Wallerian degeneration, glial scar maturation, cyst and syrinx formation, cavity formation and schwannosis. The retraction and demyelination of the spared axons may cause permanent loss of sensorimotor functions that is unresponsive to treatment [62]. Patients mostly report for cell therapy in the chronic phase of SCI. The study by Nishimura et al. recommends modification of the SCI microenvironment before cell transplantation in chronic SCI [59]. Several other studies also have been continuing to report the significance of an early intervention with cell-based therapies [63,64]. The gray area still to be understood is as to why in some patients with chronic SCI, stem cell transplantation results in marked recovery while in some it doesn’t. Perhaps, it might be due to the fact that they had incomplete SCI as a study by Liu et al. reported that the response to treatment with umbilical cord MSCs was 81.25% among incomplete SCI patients, but there was no response to treatment among the six patients with complete SCI [65]. However, the other factors need to be considered too. The limited capacity of the spinal cord to repair itself following injury is also attributed to the inhibitory factors, which are upregulated or released following injury namely the Nogo-A, chondroitin sulfate proteoglycans, semaphorin 3A and Ephrin/EphA4, that form a formidable barrier to axonal regeneration. It is worthwhile to note in this context that in addition to cell transplantation, work on Nogo-neutralization has reached the stage of clinical trials for SCI [58]. About the choice of the routes for stem cell transplantation, IT, intravenous (IV) and direct IL transplantations are the common routes employed. Samdani et al. [39] compared these delivery methods and concluded that though IL transplantation is the most efficacious, the complexity of the procedure combined with risk of damage to the already compromised tissue in 624

case of IL makes the minimally invasive IT and IV routes a better choice. Of IT and IV routes, cell engraftment and tissue sparing was better in IT compared to IV route in that study. The host immune response was also reduced in IT route. In Shin et al. study, when IL, intracisternal (IC) and IV transplantation of human bone marrow stromal cells (BMSCs) was compared, functional recovery was best in IC compared to IL and IV, while viability of the transplanted BMSCs was best in the IL group [66]. In Takahashi’s tracking of NS/PCs by bioluminescence imaging (BLI) after they were transplanted by IL, IV and IT routes, the study concluded that in terms of cell engraftment and safety, IL route was the most effective and feasible method. Further, it was also demonstrated that in the IL group even after 6 weeks of transplantation, the luminescence intensity of the grafted cells had decreased to about 10% of its initial level while in IT group, the cells were detected at the site of injury only a week later and in 6 weeks the luminescence had decreased to about 0.3% of its initial level. In the IV group, no grafted cells were detected at the site of injury [67]. Kim et al. suggested that though engraftment was higher in the IL group compared to IV group, both IL and IV transplantation of MSCs in the chronic SCI gave a significant clinical improvement. It is also significant to note that it is IL transplantation of NSCs that has shown restoration of the neuronal circuitry in a mouse model of SCI [68]. The blood--spinal cord barrier (BSCB) in SCI, which has shown to be compromised even up to 56 days after injury [69], is a limitation for IV transplantation of the cells as the cells might not reach the site in chronic SCI. Geffner et al. [70] suggested that multiple routes offered more efficacious results in cell transplantation for SCI. The dilemma still continues on how to compromise on use of IT or IV routes. These, though are minimally invasive, are only secondary to direct IL transplantation, which has associated complexities. Along with the multiple routes of stem cell injections [70], multiple injections of autologous BMSC [71] have also given favorable outcomes in patients who have no other therapeutic options. On the dosage of the cells or stem cells to be infused, it has been reported that there is a dose-dependent functional recovery after injection of BM-MSC in rats during or after the spinal shock period after SCI [72]. Still, the exact dose that needs to be injected has not been standardized. Given the multitude factors such as varied cell sources, varied levels of injury, varied routes, etc., a meta-analysis of all the studies reported till date may be of help in filling the gaps. Known-unknown IV -- vascular compromise in spinal cord injured area

2.4

During a traumatic SCI, it is not only the neurons that are affected, but also the vascular supply [73,74]. The spinal cord has a complex vasculature made up of centripetal and centrifugal circuits with terminal capillaries overlapping in a watershed region. Injury disrupts this delicate vasculature. Primary damage not only causes direct vascular disruptions but




also affects the microvasculature. The injury first affects the gray matter and then proceeds to the white matter. Apart from neural damage, deformities such as with bone fragments lead to disruption of blood supply. When there is a compromise to the blood supply in any manner, even without a physical damage to the neurons, they would suffer ischemia and undergo death if the vascular compromise continues as the neural cells are more fragile to ischemia than other cells within the body. The following secondary damage would elicit a complex response leading to the BSCB disruption and a cascade of inflammatory events [73-76]. These eventually expose the tissue around to several damage-causing substances. Once the secondary damage cascade starts, it is not clear how far there could be a chance for reversal of the damages and repair. The hypotensive status following SCI due to the spinal shock [73] further compromises the blood supply. Stem cell transplantation though has been shown to contribute to angiogenesis and vasculogenesis required for the regeneration of the damaged nerve tissue [77,78], in clinical settings, first, the damage to the vascular component is not assessed and even if assessed, the revascularization, either surgical or interventional, is not feasible because of the size of the vessels. Stabilization of the vertebral column to relieve the disfiguring compression and restoring the integrity of the vertebrae is given the first priority. The left alone vascular damage component makes the affected region handicapped in terms of nutrition and therefore the survival of the stem cells, in whatever manner applied at the site of injury, is uncertain. With the ensuing inflammation and lack of blood supply, it is doubtful whether the implanted cells such as HSCs with a capability for angiogenesis, will be able to generate an adequate blood supply for healing. On the other side, the reperfusion-related damages in such damaged and inflamed tissue is unpredictable. These gray areas may have to be thoroughly analyzed and probably an assessment of the vascular damage to the site of injury through appropriate investigations may throw light to correlate vascular compromise to the outcome. 2.5 Known-unknown V -- in vivo retention of cells to the site and their differentiation

As earlier stated, it has been identified that when a comparison is made between chronic and subacute phases of SCI, the distribution of the transplanted cells was concentrated around the lesion in the chronic SCI group due to the extensive glial scarring while it was more widely distributed in the subacute SCI group [59]. However, how long the cells or stem cells can be retained on the site of injury is an issue. Fifteen month tracking of transplanted NPCs in developing CNS has proven long-term survival maturation and differentiation into different cell types of the CNS in an animal model [79]. In a study on transplantation of green fluorescent protein (GFP) -- labeled adult NSCs in a mouse model of SCI, it was shown that the IV transplanted NSCs homed to the injury site and survived almost undifferentiated. One

week after transplantation, the NSCs retained their proliferation potential and could form neurospheres when recovered from the lesion and cultured in vitro [42]. Even so, the situation is different clinically wherein retention of the transplanted cells to the site of injury still remains a problem, for which various biomaterials offer a potential solution [80]. Tissue engineering employing scaffolds is an attractive option for optimal in vivo repair in SCI. Scaffolds like collagen, poly lactic-co-glycolic acid (PLGA), PEG, etc have been employed for cell transplantation in SCI. In a study there was significant improvement in limb function when Type I collagen with soluble Nogo receptor, chondroitinase ABC, and MSCs were transplanted into the hemi-resection defect in the rat spinal cord but this improvement was not observed in the control group without scaffold [81]. Among the scaffolds for SCI, hydrogels that are three-dimensional (3D) networks of hydrophilic polymers that are held in place by covalent bonds or other cohesive forces are considered very attractive biomaterials due to their tissue-like mechanical abilities, porous structure, ability to incorporate growth promoting/adhesion molecules to enhance cell attachment and tissue growth and capacity to incorporate drug/gene vectors for targeted delivery [82]. A study that employed PLGA scaffolds with NSC in canine SCI reported that the PLGA bridged the tissue defects and there was nice integration of the NSCs in the PLGA with the residual canine spinal cord tissue. Further ectopic expression of a therapeutic neurotrophin-3 gene could also be possible in the NSC seeded within the PLGA scaffolds [83]. Studies employing these scaffolds have proven the survival of cells after transplantation, integration of the cells with the residual spinal cord tissue, differentiation into neurons and even synapse formation [81-85]. Scaffolds also can be modified appropriately to promote lineage-specific differentiation. For instance, a hydrogel blend of hyaluronan and methyl cellulose modified with recombinant rat platelet-derived growth factor-A has been employed to promote oligodendrocytic differentiation of adult brain-derived NS/PCs and in SCI rats these cells transplanted with the modified hydrogel scaffold promoted better behavioral recovery than in the rats where NSPCs were transplanted in media [86]. Similarly, collagen scaffolds incorporated with cetuximab has been shown to enhance neuronal differentiation and inhibit astrocytic differentiation [87]. Even altering the scaffold diameter has shown to modify the environment to favor axon regeneration [88]. In addition to scaffolds, another major area of interest has been on the study of molecules to improvise SCI repair. These molecules under study include the CNS inhibitors, which may be the canonical axon guidance molecules (e.g., semaphorins, ephrins, netrins), prototypic myelin inhibitors (Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein) or chondroitin sulfate proteoglycans (lecticans, neuron-glial antigen 2) [89]. By appropriately including or eliminating these molecules in scaffolds, designing strategies for successful SCI repairs are being undertaken. The molecules that promote neuronal growth and sprouting like the


625



extracellular matrix molecules, cell adhesion molecules and neurotrophic factors are also being explored [89]. Osanai et al. in their study on surgical transplantation of BMSCs embedded in a TGP scaffolds onto the intact neocortex in infarct brains of mice concluded that there were relatively more neural precursors in this group compared to the one where BMSCs were transplanted alone without TGP [85]. Predominantly, these 3D approaches are giving better outcomes when used as substrates to provide controlled release of neurotrophic factors to the injured site [85,90-92]. There have not been any clinical trials employing scaffolds for cell therapy in SCI. This is because the gray area is to identify the most suitable scaffold that does not have toxicity, is readily implantable, porous to allow diffusion of nutrition, supports angiogenesis to the needed extent, supports cell proliferation, will direct the cell differentiation in the desired direction, biodegradable, does not have the risk of biological contamination with animal proteins, non-teratogenic or non-mutagenic, freely detectable in appropriate diagnostic modalities and accessible. Known-unknown VI -- synapses formation mechanisms after stem cell application in spinal cord

2.6

Remyelination and synapse formation after stem cell transplantation are important mechanisms underlying recovery in SCI. Histological studies in dogs posttransplantation of hUCB-derived MSCs has demonstrated remyelination of axons by P0-positive myelin sheath [33]. In a rat toxin-induced model of spinal cord demyelination, when remyelination by transplantation of OECs from embryonic, neonatal and adult rats were compared, it was identified that the process was more efficiently achieved by embryonic-derived OECs [93]. In another study on transplantation of NS/PCs in myelindeficient shiverer mutant mice, it was proven than remyelination by the graft-derived oligodendrocytes contributes greatly to the functional recovery after SCI [94]. Synapse formation after cell transplantation in SCI has been demonstrated by Abematsu et al. Their study indicated that transplantation of NSCs along with administration of valproic acid in a mouse model of SCI resulted in restoration of hind limb function with the transplant-derived neurons reconstructing the broken neuronal circuits and forming functional synaptic connections with endogenous neurons as revealed by appropriate scientific methods [95]. Ben-Hur in his commentary on the Abematsu et al.’s article questioned whether these findings would be pertinent to human patients because of the outlined differences between animal models and human patients in terms of the anatomy and function of the spinal cord [96]. He states that in humans, gait and many other voluntary limb motor functions are relatively more dependent on the corticospinal tract than in rodents, and hence the question of whether the findings of Abematsu et al. will be significant to humans can be answered only if it is identified whether the transplanted stem cells establish synapses preferentially with certain tracts. 626

Also, synapse formation mechanisms depend on the type of cell source employed. Parr et al. showed that MSCs from the BM showed no neuronal differentiation, while NS/PCs derived from the ependymal region of the spinal cord demon-strated astrocytes and oligodendrocytes differentiation. The study hypothesized that the beneficial mechanisms of action in SCI with regard to the MSCs could be mainly by the potential axonal guidance through the guiding strands of matrix generated by these MSCs [97]. Moreover, even if neuronal differentiation of transplanted cells is achieved, whether these transplanted stem cells will help in establishment of exact neuronal circuitry with the higher CNS that was present before the injury remains unanswered. When it comes to regeneration after cell transplantation in the SCI and CNS injury, the anatomical differences need to be considered [95]. In vitro, cell culture technologies though similar for both SCI and CNS cell therapy, the secondary cascade of inflammatory events in SCI along with axonal regeneration being the only modality of nerve regeneration in most SCI cases along with progressive cell death in CNS [98] give rise to the need for development of tailored cell therapy strategies separately for SCI and CNS repair. This makes us understand that simple nerve repair and synapse formation demonstrated in vitro and in vivo laboratory models may not be sufficient for a successful outcome in human patients where the mode of synapse formation with the various tracts in the spinal cord and with the higher CNS is different. To enhance the functional recovery in SCI, neuroprosthetic technologies like functional electrical stimulation, intracortical microstimulation, electroencephalography-based brain--computer interfaces [24] are being employed. In cultured cardiac constructs in vitro, it has been proven that application of electric signal stimulation induces cell alignment, coupling, increases the synchronous contraction and helps in good ultrastructural organization [99]. So, we hypothesize that perhaps such electric stimulation of the cells or stem cells in vitro before transplantation in SCI or after transplantation may aid in proper reorganization of neurons while increasing their ability to establish functional circuitry. This thought needs to be validated by appropriate scientific studies in which a combination of electrical stimulation in optimal dosages and relevant frequencies could be evaluated. Known-unknown VII -- long-term tracking of implanted stem cells

2.7

In vivo tracking of stem cells is essential to monitor their survival, migration, therapeutic response and to identify the transplantation parameters that are advantageous for optimal cell/tissue regeneration. An ideal tracking method must employ agents, molecules and devices that are non-toxic to humans, does not induce genetic modification in the cells, preferably non-invasive, biocompatible, provide high resolution, are highly specific, sensitive, have the ability to track the stem cells as they travel to different sites in the body, offer



Synapses in brain vs SC

Animal models vs Human gap omical variation between Anat the two

Arena for research

Animals are young

Study on all possible

Humans have

physical/chemical stimuli

co-morbidities

to regenerate lost synapses

Time, Route and Dosage

Synapse regeneration differ between brain and SC

WHEN? to apply cells


WHICH route? Reach to site and retention

Intra: thecal or lesional or venous How much cells?

Applied cells do they reach

Vascular compromise

Find safe scaffolds

and retain

...is difficult to

to retain the cells in vivo

at site of injury?

diagnose.

Arena for research

and support regeneration

even if diagnosed revascularization Source of cells

is difficult

WHICH is the best?

Long term tracking Arena for research Develop a safe in vivo cell -labelling/tracking technology

iPS, ES

A non-invasive in vivo tracking of

Foetal SC

the cells for a long

Cord blood SC Auto. BMSC

is not available

Figure 1. The figure depicts the seven categories of ‘known-unknowns’ in the application of stem cells in spinal cord injury along with the thrust areas for future research. BMSC: Bone marrow stromal cell.

real-time visualization and should have the ability to identify the state of stem cell differentiation [100]. Tracking of stem cells in vivo is usually done by employing methods like MRI, nuclear medicine imaging and optical imaging [100,101]. MRI involves labeling of the stem cells with a contrast agent to produce a positive or negative signal so that the cells can be distinguished from the background. Contrast agents include paramagnetic (gadolinium-based or manganese-based) or superparamagnetic (iron oxide-based) agents. Superparamagnetic iron oxide (SPIO) is the widely studied agent and it is approved by the US FDA for use in humans [101]. MRI using SPIO has been used in clinical studies also. Zhu et al. reported implantation of NSCs labeled with SPIO in a patient with traumatic brain injury. The patient was followed up with T2-weighted MRI every week for 10 weeks. The results showed that the hypointense signal generated by the cells showing the movement of the cells from the implantation site to the lesion’s periphery. However, this hypointense signal disappeared by the 7th week, which the authors attributed to the proliferation of NSCs [102]. The

disadvantage with SPIO is the risk of long-term health problems due to endocytosis of these magnetic particles with CNS [100,103]. In addition, the labeling of stem cells with these SPIOs employs toxic transfection agents. To overcome this, citrate-coated SPIOs have been reported [103]. Nuclear medicine imaging techniques include single photon-emission computed tomography (SPECT) and positron emission tomography (PET). In nuclear medicine imaging, imaging radiotracers, which can bind to different ligands, are employed. This modality is extremely sensitive and even nanomolecular level details can be visualized. Nuclear imaging involves direct and indirect imaging. Direct imaging involves labeling the cells directly with a radioactive probe. However, the disadvantages include the ‘leakage of the radionuclides into non-target cells, limited time window for imaging due to half-life decay, dilution of signal from cell division, and lack of ability to determine cell viability and function.’ Indirect imaging involves the use of reporter genes. As these reporter genes are passed onto the progeny, long-term tracking is possible. Enzyme-based, receptor-based and


627



transporter-based gene imaging systems have been reported [101]. Optical imaging includes fluorescence imaging (FLI) and BLI. FLI employs organic fluorophores, which are fluorescent dyes such as DiD, Dil and indocyanine green (ICG). ICG is FDA approved. Endogenous fluorescent proteins like GFP and red fluorescent protein are also used. Biocompatible inorganic fluorescent semiconductor nanocrystals called quantum dots (QDs) are also being used for in vivo stem cell tracking [104]. QDs have disadvantages like large size (10 -- 30 nm) and their ‘blinking’ behavior may lead to dark periods of no emission interrupting longer periods of fluorescence [104]. BLI involves transducing a reporter gene into the stem cells that codes for firefly luciferase or renilla luciferase. This modality is useful for long-term tracking and cell quantification, but the risks of engineering the cell thereby transferring unwanted mutations make it less advantageous for clinical translation [101]. In a meta-analysis, it was observed that 19 human therapeutic trials have been reported employing 18-fluorodeoxyglucose-PET, 111-indium-SPECT; 99-technetium-SPECT, and iron oxide-MRI tracking methods for stem cells for different diseases like myocardial infarction; Chagasic cardiomyopathy; ischemic stroke; traumatic injury of brain or spinal cord; diabetes and cirrhosis [105]. But none of these methods fulfill all the requirements for successful clinical application [100]. Thus, an ideal tracking method needs to be identified, one that can track the stem cells for longer periods of time in vivo to study the factors such as cell integration synapses formation and tissue repair. Li et al.’s postulation of a biological global positioning system using a combination of genetically labeled stem cells and in vivo imaging technology [100] may be a crucial step for progress in the arena of stem cell tracking. 3.

Conclusion

Several clinical trials have been undertaken aiming at establishing safety and efficacy of cell or stem cell-based therapies in SCI. However, the expected clinical outcomes have not been reached yet, given the multitude of known-unknown factors that have not yet been explored to the fullest. Thus, in this review, the various ‘known-unknowns’ in SCI repair after cell transplantation have been briefly dealt with, followed by suggestions to overcome these gray areas, making this a valuable guideline for focusing on thrust areas of research on the seven different areas mentioned and developing protocols for optimal spinal cord regeneration. 4.

Expert opinion

A critical analysis of the seven categories of ‘knownunknowns’ reveals that the cell-based therapies in both animal models and human victims of spinal cord injuries have been safe as of now and the way forward is to undertake a meta-analysis of the facts available at the moment and give 628

priority to areas, which are likely to yield potential findings in order to fill the gap between the objectives and reality. It has become clear that the area of interest, that is, SCI is of paramount importance given the background of young victims, social, financial and other liabilities and above all the quality of life that should be improved and with no definitive therapies at the moment. The goal being, neural regeneration and functional circuitry formation, we give hereunder two major areas for thrust research in the descending order of importance, from our view, followed by two minor areas of research. The two major areas are: 1) The retention of transplanted cells at the site of injury is an arena that has not been studied much, and therefore this needs a thorough analysis. Combining the findings on the timing of application of the cells at the site of injury and the mode of application, it is advisable that the studies in animal models and human victims be conducted with IL application of scaffoldimpregnated autologous stem cells, which might be advantageous. 2) Basic studies and animal studies should be undertaken to find potential stimulants for neural circuitry formation. Electric stimuli could be one among them. As electric stimulation of the brain in Parkinson patients has reached the level of clinical studies [106], a combination of both electrical and chemical stimuli is worth investigating so that such stimuli once found safe and efficacious in basic studies can be translated to human studies, which might yield better results along with cell-based therapies. 3) A safe, non-cytotoxic, preferably bio-contamination free non-invasive tracking of the in vivo implanted cells or stem cells is equally important that enables us to track the physical movement of the cells and to study their retention over longer periods of time, to correlate the same with clinical findings. The two minor areas of evaluation: 1) A meta-analysis of all the data based on the seven known-unknown from both animal and human studies be undertaken to fill the gaps. 2) From the cell differentiation point of view, an analysis from the studies with various types of cells starting from ESCs, iPSCs and other pluripotent stem cells is essential, which, after establishing safety parameters, can be combined with the above-mentioned major areas of research are likely to yield the appropriate scaffold for implantation and retention as well as additional physical and chemical stimuli to form functional circuitry. Figure 1 depicts the seven categories of ‘known-unknowns’ along with the thrust areas for future research.




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Affiliation Vidyasagar Devaprasad Dedeepiya1,2 MD DNB, Justin Benjamin William3 MVSc PhD, Jutty KBC Parthiban1 MCh (Neurosurgery), Ranganathan Chidambaram4 MD, Madasamy Balamurugan4 MD, Satoshi Kuroda5 MD PhD, Masaru Iwasaki6 MD PhD, Senthilkumar Preethy7,8 BDS & Samuel JK Abraham†1,6 MD PhD † Author for correspondence 1 Nichi-In Centre for Regenerative Medicine (NCRM), The Mary-Yoshio Translational Hexagon (MYTH), PB 1262, Chennai -- 600034, Tamil Nadu, India Fax: +91 44 24732186; E-mail: [email protected], [email protected] 2 Acharya Nagarjuna University, Department of Biotechnology, Guntur, India 3 Madras Veterinary College, Department of Surgery and Radiology, Chennai, India 4 Ruma Biotherapy and Research Centre, Chennai, India 5 Toyama University- Graduate School of Medicine, Department of Neurosurgery, Toyama, Japan 6 Yamanashi University, Faculty of Medicine, Chuo, Japan 7 Nichi-In Centre for Regenerative Medicine (NCRM), The Fujio-Eiji Academic Terrain (FEAT), Chennai, India 8 Hope Foundation (Trust), Chennai, India

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Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms.

Spinal diffusion tensor imaging: a comprehensive review with emphasis on spinal cord anatomy and clinical applications.

Infertility in men with spinal cord injury: research and treatment.

Induced Pluripotent Stem Cell Therapies for Cervical Spinal Cord Injury.

Sexuality for women with spinal cord injury.

Development of a database for translational spinal cord injury research.

Spinal cord injury models: a review.

Sleeping with spinal cord injury.

Effects of vibration on spasticity in individuals with spinal cord injury: a scoping systematic review.

Hydrogels and Cell Based Therapies in Spinal Cord Injury Regeneration.

Screening for neuropathic pain after spinal cord injury with the spinal cord injury pain instrument (SCIPI): a preliminary validation study.

Toward a health services research capacity in spinal cord injury.

Urolithiasis in children with spinal cord injury.

Osteoporosis in individuals with spinal cord injury.

Electrospun Fibers for Spinal Cord Injury Research and Regeneration.

Protocol for a scoping review of skin self-care of people with spinal cord injury.

Cochrane in CORR1: Steroids for Acute Spinal Cord Injury (Review).

Short Form health surveys and related variants in spinal cord injury research: a systematic review.

Interventions for improving employment outcomes among individuals with spinal cord injury: a systematic review.

Screening for bladder cancer in individuals with spinal cord injury.

Scales on Quality of Life in patients with spinal cord injury: integrative review.

The older adult with a spinal cord injury.

Review article: post-spinal cord injury syringomyelia.

Autonomic hyperreflexia with spinal cord injury.