|Functional Recovery of Spinal Cord Injury Following Application of
Intralesional Bone Marrow Mononuclear Cells Embedded in Polymer
Scaffold - Two Year Follow-up in a Canine
|Justin Benjamin William1, Rajamanickam Prabakaran1, Subbu Ayyappan1, Haridass Puskhinraj1, Dhananjaya Rao1, Sadananda Rao
Manjunath2, Paramasivam Thamaraikannan2, Vidyasagar Devaprasad Dedeepiya2, Satoshi Kuroda3, Hiroshi Yoshioka4, Yuichi Mori4,
Senthilkumar Preethy2,5 and Samuel JK Abraham2,6*
|1Madras Veterinary College, Chennai, India
|2Nichi-In Centre for Regenerative Medicine, Chennai, India
|3Department of Neurosurgery, Hokkaido University- Graduate School of Medicine, Sapporo, Japan
|4Waseda University, Tokyo, Japan
|5Hope Foundation (Trust), Chennai, India
|6Yamanashi University- Faculty of Medicine, Chuo, Japan
||Dr. Samuel JK Abraham
Nichi-In Centre for
C 16 &17, Vijaya Health Centre Premises 175, NSK Salai
Vadapalani, Chennai – 600026, India
Tel: +91-44-42321322 / 24816743
E-mail: email@example.com, firstname.lastname@example.org
|Received November 05, 2011; Accepted November 19, 2011; Published November 21, 2011
|Citation: William JB, Prabakaran R, Ayyappan S, Puskhinraj H, Rao D, et
al. (2011) Functional Recovery of Spinal Cord Injury Following Application of
Intralesional Bone Marrow Mononuclear Cells Embedded in Polymer Scaffold -
Two Year Follow-up in a Canine. J Stem Cell Res Ther 1:110. doi:10.4172/2157-
|Copyright: © 2011 William JB, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
|Background: Bone marrow derived pluripotent stem cells hold a great promise for therapeutic repair of injured
central nervous system. This report is on a six- month old paraplegic Boxer breed canine with traumatic spinal cord
injury at the level of T12, which functionally recovered following intralesional transplantation of autologous Bone
Marrow Mono Nuclear Cells (BMMNCs) seeded on a Thermoreversible gelation polymer (TGP) combined with
intravenous Cell Transplantation.
|Materials and Methods: Thirty ml of Bone Marrow was aspirated and BMMNCs were isolated. From the total
BMMNCs isolated, 20 x 106 cells were seeded in 1.5 ml of TGP and implanted at the site of injured spinal cord. A
fraction of BMMNCs isolated were stored at -80°C from which 4.16 x 106 BMMNCs were thawed and transfused
intravenously by suspending in 2ml saline on the 19th post-operative day. The animal was followed up by assessment
every two weeks for a period of two years.
|Results: Recovery of motor and sensory functions were noticed on the 53rd day, attempt for standing on the
79th day and ambulation on the 98th day after the initial cell transplantation. The animal had satisfactory ambulation
on the 133rd day and thereafter the life style of the animal was gradually restored to normalcy. Status quo of this
recovery has been maintained for the past two years.
|Conclusion: The outcome proves the safety of intralesional transplantation of autologous BMMNCs embedded
in TGP in spinal cord injury and makes us recommend the same for more number of similar cases.
|Bone Marrow Stem cells; Canine Diseases; Cell
Transplantation; Spinal Cord Injuries; Thermoreversible Gelation
|TGP- Thermoreversible Gelation Polymer; SCISpinal
Cord Injury; BMSC- Bone Marrow Stromal Cells; BMMNCs-
Bone Marrow Mononuclear Cells; LAL- Limulus Amebocyte Lysate;
FITC- Fluorescein Isothiocyanate; SEP- Somatosensory Evoked
Potential; HSC- Hematopoietic Stem Cells; MSC-Mesenchymal Stem
|Regenerative potential of central nervous system is limited  and
treatment of traumatic Spinal Cord Injury (SCI) in canines continues
to be a challenging task. SCI leads to severe functional impairment like
paraplegia, quadriplegia and tetraplegia with upper or lower motor
neuronal deficits and causes severe distress, devastating changes in
quality and life expectancy of the animal, with a frustrating situation
to the pet owners. Functional deficits following SCI result from
interruption in axonal tracts or damage to axons, loss of neurons,
oligodendrocytes, astrocytes, endothelial cells, precursor cells and
demyelination . With the initial mechanical insult to the spinal
cord, SCI also leads to a series of secondary cascades like ischemia,
anoxia and free-radical formation, which impede regeneration of
axons due to the release of myelin associated inhibitory proteins,
extracellular matrix-derived inhibitory cues and glial scar formation [3,4]. The key elements of repair of SCI require not only the neural cell
proliferation and survival, but also the promotion of axonal growth,
remyelination and neosynaptogenesis . The treatment attempts
on spinal stabilization by surgical procedures mainly aid to restore
the anatomical integrity of the damaged vertebrae and prevent the
secondary cascade of events without any therapeutic potential for
spinal cord regeneration . Stem cell-based transplantation therapies
are being attempted as the current regenerative pathway for the
treatment of spinal cord lesions and several animal experiments as well
as clinical trials are being reported to promote neuronal regeneration
and improve spinal cord function [7-9]. Bone Marrow Stromal Cells (BMSCs) and Bone Marrow Mononuclear Cells (BMMNCs) in animal
model studies of SCI have been found to replace white and grey matter,
neuronal and axonal regeneration, astrocyte proliferation, myelination,
neovascularisation and functional improvement which presents an
encouraging scope for clinical translation [10,11]. Further engraftment
of stem cells with biomaterial scaffolds provides a promising strategy
for engineering diseased tissues and cellular delivery. Numerous
previous studies have used a variety of natural (e.g., collagen, fibrin,
chitosan, agarose, and alginates) and synthetic (e.g., Poly (lactic-coglycolic
acid), Poly (ethylene glycol), poly (N-isopropylacrylamideco-
n-butyl methacrylate), copper capillary alginate gel) polymers for
repair of damaged spinal cord or brain [12-16]. Thermo reversible
gelation polymer (TGP), a temperature-dependant visco elastic
synthetic scaffold has been reported to promote in- vitro 3D culture
of cells and tissues in hydrogel state at 37ºC and also aid the tissue
regeneration process by activation of stem cells and prevention of the
inflammatory process . Several in- vitro and animal model studies
also demonstrate that TGP promoted regeneration of damaged tissues
like pancreas , liver , cornea , and neural tissues . Very
recent studies have reported that surgical transplantation of TGP
constructed bone marrow-derived stem cells enhance the engraftment
of donor cells onto the cerebral infarct of mouse neocortex [21,22].
|However, knowledge on intralesional application of BMMNC
seeded in TGP and intravenous administration of BMMNCs for
traumatic SCI in canines is limited. Here, we report our results after a
two-year follow-up that along with decompressive surgical procedure,
transplantation of autologous BMMNC seeded with TGP applied
intralesionaly and intravenously can aid in functional recovery of
traumatic SCI in canines.
|The case report
|Patient history: A six month old, congenitally deaf, intact male
Boxer cross-bred canine with body weight of 15 kilograms was brought
to our hospital for treatment of paraplegia with total loss of motor and
sensory functions of the hind limbs and that of bladder and bowel
function. The canine was brought four days after an automobile accident
when it was on a loose-leash walk on the road. The animal was found to
be comfortable in sternal recumbency posture and showed severe pain
on palpation at caudal thoracic region. Distended bladder with absence
of mictiruition and defecation was noticed and the vital signs were
within the clinical limits. Deep pain reflex, conscious proprioception
reflex, patellar reflex of the hind limbs were absent and the panniculus
reflex was normal up to the level of T10 vertebra with a decrease on the
right side and absence on the left side at T11 vertebra and caudal to it.
Anal sphincter reflex was intact. Neurological examination of the above
spinal reflexes was indicative of upper motor neuron lesion and was
localized between T10-13 vertebrae. The clinical severity of the neural
injury was classified as per Denny and Butterworth  as Grade 5
paraplegia, and the pelvic limb function, according to Olby et al. 
scoring system, was scored as stage 1 and point 0 condition (Figure
1A). The owner was counseled and an informed consent was obtained
to perform the decompressive surgery with autologous bone marrow
stem cell therapy. Approval for the study was obtained from the ethics
committee of the Madras Veterinary College in which the study was
conducted. All procedures were accomplished in accordance with the
national and international Guidelines for Care and Use of animals in
scientific research. The canine was induced anaesthesia with propofol
at the dose of 4.5 mg/kg B.W. and maintained under isoflurane as inhalant agent throughout the procedures which were performed on
the same day.
||Figure 1: Caption: Pre-operative, Intra and Post-operative Images of the
Canine with spinal cord injury treated with autologous Bone marrow stem cells.
|Localization of lesion: Plain radiography followed by myelography
using Iohexol (Omnipaque, 350 mgI/ml @ 0.3 ml/kg body weight
intracisternally) revealed compression fracture of the 12th thoracic
vertebra and abrupt stoppage of the contrast column cranial to 12th
thoracic vertebra. (Figure 1B)
|Bone marrow Aspiration: Right femur was prepared and using
Jamshidi needle, 30 ml of bone marrow was aspirated under C-arm
guidance (Figure 1C). The bone marrow was preserved in a bag
containing citrate dextrose anticoagulant and transported in cold chain
storage (4° to 8° C) to a Central cell processing facility.
|Processing of BMMNCs & Preparation of TGP Construct: The
aspirate was processed under cGMP SOP’s Class 10000 clean room and
class 100 bio-safety hood. BMMNCs were isolated using Ficoll gradient
method and were counted using Neubaur’s hemocytometer. From the
total quantity of BMMNCs isolated, 20 x 106 cells were seeded in 1.5 ml
of thermoreversible gelation polymer(TGP) which is a copolymer composed
of thermoresponsive polymer block [poly(N-isopropylacrylamide-
co-n-butyl methacrylate)(poly(NIPAAm-co-BMA)] and the hydrophilic polymer block (polyethylene glycol [PEG]). A fraction of
the cells was preserved in – 80°C.
|Quality control testing: Before seeding the cells in the TGP, the
cells were subjected to Flowcytometry analysis for quantifying the
CD34+/CD45- cells by appropriate fluorescein isothiocyanate (FITC)
antibodies (Becton Dickinson, Jan Jose, USA) and analyzing data’s
using BD Cell quest pro software. The Endotoxin test was also carried
out using Limulus Amebocyte Lysate (LAL) Kit method for confirming
the sterility before cell transplantation.
|Hemilaminectomy and Intralesional Engraftment: Left
hemilaminectomy was performed as per the technique described
by Wheeler and Sharp  and durotomy at T12 vertebra and the
injured spinal cord was exposed. The injured site was oedematous and
durotomy revealed blood clots (Figure 1D). The construct of 1.5 ml
TGP seeded with 20 x 106 BMMNCs was engrafted in liquid phase at
the site of injured spinal cord and within a few minutes the construct
became solidified. The laminectomy defect was overlayed with fat graft
harvested from the subcutaneous tissue and the surgical site was closed
in a routine fashion. No stainless steel metallic implants were used for
internal fixation of the vertebral fracture due to the concern on postoperative
|Intravenous Transfusion: On the 19th postoperative day, 4.16
x 106BMMNCs were thawed from previously stored BMMNCs and
suspended in 2 ml of normal saline and transfused intravenously.
|Post-operative management included antibiotics, analgesics,
bladder management, nursing care to prevent decubital ulcers, cage
rest for six weeks and passive range of motion- physiotherapy. Motor
and sensory functions were evaluated every two weeks post-operatively
and Olby scoring system  was used for quantitative evaluation of
functional outcome in this study.
|Post-operatively after intralesional BMMNC transplantation no
toxic and adverse effects were noticed. The canine was monitored
continuously for the first 72 hours and no alterations in vital sign
parameters were recorded. The animal was discharged from the
hospital on the fifth day and the owners were explained throughouly
on the follow-up care and management strategies. On the 14th day,
seroma formation at the surgical site was noticed and the serous fluid
was tapped out. Also, slight deep pain sensation and tail wagging
were noticed. Towel sling exercise was advised to be followed until
the recovery of unassisted standing and on the 16th day, a slight dorsal
elevation of the vertebra at the surgical site was noticed. It indicated a
slight disruption in the vertebral alignment. In order to potentiate the
efficacy of cell transplantation as reported earlier  a second dose
was administered on the 19th day by intravenous route and no adverse
reactions were noticed following the same. Neurological examination
on the 28th day revealed a return of deep pain sensation in its right hind
limb and a slight sluggish movement in the left hind limb. Involuntary
movement of the right hind limb and absence of movement in the
left hind limb was noticed. Defecation was reported to be normal and
bladder function was maintained by manual evacuation by the owner.
On the 42nd day, improvement in deep pain, patellar reflex, involuntary
movement and conscious proprioception reflex was noticed in both the
hind limbs and panniculus reflex was noticed caudal to T12 vertebrae.
Transient unassisted standing on the hind limbs was noticed on the 53rd day (Figure 1E). The animal was encouraged to move freely and it made
transient self attempts to stand by itself on the 79th day. Thereafter, the
animal made attempts to stand and walk with in-coordinated hind limb
movements. It made ambulation with in-coordinated left hind limb
movement on the 98th day (Figure 1F) and on the 133rd day unassisted
standing, normal ambulation and ability for prolonged walking
resumed and a normal life style of the animal was fully restored. Longterm
follow up on the 180th day confirmed that the animal continued its
normal life style with normal pelvic gait movements with no recurrence
of neurological disorders. Subsequently, the animal was followed up
periodically and the last follow up was at the end of two years since
the first cell transplantation, which confirmed the status quo of all the
improvements upto the restoration of normalcy. The Olby scores of
pelvic limb status from the postoperative period to the recovery time
are listed in Table 1.
||Table 1: Assessment of functional outcome based on pelvic limb function by Olby
|In canines, the treatment strategies for complete recovery from
traumatic spinal cord injury are still under research. Till date, irrespective
of the type of strategy followed, treatment for severe SCI remains to be
unresolved. The current clinical and animal studies on treatment of SCI
aim to inhibit the secondary inflammatory and degenerative changes
and to augment the neural regeneration. Reports reveal that stem cells
transplanted into the injured lesion differentiate into oligodendrocytes
and astrocytes, integrate into axonal pathways and regenerate and
remyelinate the injured axons [27-30]. BMMNCs of autologous origin
offer advantages of multi-potency with definitive in- vivo and in- vitro
neuronal differentiation, avoidance of immunological and ethical
issues. In murine model studies, BMSCs after transplantation are
demonstrated to migrate and attach with the injured neural tissue and
the cells finally disappeared within three weeks indicating the release
of some trophic factors from BMSCs to rescue neurons and glial cells
from degeneration and to stimulate differentiation of neural stem cells
in the injured spinal cord tissue [6,31,32] Similar mechanisms are
expected on intralesional and intravenous route of administration of
BMMNCs where the cells could possibly migrate to injured spinal cord
tissue and repair the damaged tissue as reported earlier . Though
BMSCs present an attractive strategy, their purification and expansion
is quite a cumbersome process. In contrast, BMMNC isolation is
relatively easy. Importantly, BMMNC contains different cell fractions including CD34+ Hematopoietic stem cells (HSC), Mesenchymal stem
cells (MSCs) and endothelial progenitors. In principle, organogenesis
or tissue regeneration by any type of cell therapy should go hand
in hand with angiogenesis, where the tissue building process as
it progresses should be supported by blood supply for successful
regeneration of the damaged or dysfunctional organ. Studies have
proven that the application of whole BMMNCs is more successful than
methods which use sub fractionated cell preparations . In a study
when transplantation of human BMSCs and BMMNCs into rats with
SCI was compared, it was observed that BMMNCs did not give rise to
mature immune cells after transplantation which is a common issue
concerned with allogeneic BMMNC transplantation. There was no
increased host immune response or tissue loss when compared with
BMSC-transplanted animals. In contrast there was an increased host
macrophage/microglia response after BMSC transplantation which
the authors attributed to exposure of cells to serum-containing media.
The efficacy of BMSCs and BMMNCs were found to be similar in that
study . In our study since the animal is alive, we are unable to
show the post-transplantation pathology of the spinal cord, which is
a limitation. As per the evidences pointed out earlier, the application
of BMMNCs is justified as they are relatively safe, easy to obtain and
have proven efficacy in treating SCI. The temperature-dependant solid
and liquid phase properties of TGP scaffold material helped to engraft
the stem cells in gel phase of TGP over the injured spinal cord area
exposed through the laminectomy defect and after the solidification,
the TGP – stem cell construct was observed to be intact without loss
of structural stability. Further, TGP could have favoured neuronal
and oligodentrocyte differentiation of residential neural stem cells and
the BMMNCs. Post-operatively, during recovery period, slight dorsal
elevation of the thoracic vertebrae secondary to malalignment of the
injured vertebrae was noticed. This could be due to the lack of adequate
epaxial muscle support following surgical trauma, inadequate muscle
strength and support of the hind limbs to maintain the posture and
absence of internal fixation at T12 vertebral segment and these reasons
could also be attributed to the delayed recovery. In this case study,
assessment of the severity of SCI by somatosensory evoked potential
(SEP) values and its correlation with the functional outcome was
not carried out and it is reported that the functional scoring system
was found to be more sensitive than SEP measurements . For the
quantitative assessment of the functional outcome of the Canine SCI
based on the pelvic limb function, Olby scoring system  was used
and the reliability of the same has been confirmed by previous reports
[35,36]. During the post-operative follow up period, when the canine
was left on Marble and Granite floors which were slippery, the animal
had difficulty in initiating attempts to stand by itself which could also
be attributed to the delay in recovery of pelvic limb functions. Later,
covering the floor with carpet or non-slippery floor conditions helped
the canine to make more progressive attempts to stand on its own.
In canines, interestingly, few cases have been reported to be able to
walk following severe spinal injury, but they fail to regain deep pain
and continence due to higher input from higher center mediated
through a few intact axons surviving across the lesion and is termed as
spinal reflex walk. These canines always remained incontinent and the
recovery of walking ability occurred only after four months . In this
case study, a full functional recovery was noticed after three months
and the recovery has been sustaining for more than two years. These
results indicate that a combination of surgical decompression with
intralesional transplantation of BMMNCs seeded in TGP followed
by an intravenous injection could produce a functional recovery of injured spinal cord in canines. However, the fate of the transplanted
cells remains to be investigated in the regeneration of the spinal cord
after an injury.
|Our clinical study revealed that the intralesional implantation of
autologous BMMNCs seeded in a TGP followed by an intravenous
injection into a canine was safe without any complications following
the treatment for two years. The functional recovery could be due to
the beneficial effects of BMMNCs combined with the decompressive
procedures as they might have helped to hasten neurological recovery,
which otherwise could not have been expected in this short-span, going
by the earlier reports. The factors determining the outcome could be
the age of the canine, severity of the injury, time interval between the
injury and the cell transplantation, mode of transplantation, role and
utility of TGP scaffold and the dosage of the stem cells transplanted, all
of which need to be evaluated elaborately. Further in-vitro and in-vivo
studies are needed to clarify the mechanisms of action of BMMNC on
neuronal regeneration to confirm the above results. Though safety of
the procedure has been proven in this case, a larger study is warranted
to ascertain the efficacy.
|The authors acknowledge M/S Hope Foundation (Trust), Chennai, India, for
funding the study and Mr. V. Sampathkumar for technical assistance.
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