Objectives: To explore the clinical therapeutic effects and safety of autologous
bone marrow mesenchymal stem cell therapy for traumatic brain injury by lumbar
Materials and Methods: A total of 97 patients
(24 with persistent vegetative state and 73 with disturbance motor activity) who
developed a complex cerebral lesion after traumatic brain injury received
autologous bone marrow mesenchymal stem cell therapy voluntarily. The stem cells
were isolated from the bone marrow of the patients and transplanted into the
subarachnoid space by lumbar puncture.
Results: Fourteen days after cell therapy, no serious complications or adverse
events were reported. To a certain extent, 38 of 97 patients (39.2%) improved in
the function of brain after transplant (P = .007). Eleven of 24 patients (45.8%)
with persistent vegetative state showed posttherapeutic improvements in
consciousness (P = .024). Twenty-seven of 73 patients (37.0%) with a motor
disorder began to show improvements in motor functions
(P = .025). The age of patients and the time elapsed between injury and therapy
had effects on the outcomes of the cellular therapy (P < .05). No correlation
was found between the number of cell injections and improvements (P > .05).
Conclusions: This study suggests that the bone marrow stem cell therapy is safe
and effective on patients with traumatic brain injury complications, such as
persistent vegetative state and motor disorder, through lumbar puncture. Young
patients improve more easily than older ones. The earlier the cellular therapy
begins in the subacute stage of traumatic brain injury, the better the results.
Key words: Cerebral lesion, Regenerative therapy, Transplant, Vegetative state, Motor.
It is well known that traumatic brain injury (TBI) is a major public health
problem worldwide. Traumatic brain injury is the No. 1 cause of coma and the
leading role in disability in children and young adults.1 Recently, prehospital
and intensive care of patients with TBI has improved substantially, and
evidence-based guidelines for management have been developed.2 However, the
prognosis for patients with severe TBI remains poor, such as disturbance of
consciousness and motor disorder. Even under the best circumstances, mortality
for acute severe TBI is around 36%, it is 15% for severe disability, it is 20%
for moderate disability, and 25% for complete recovery.3 Because the
regeneration capacity of neurons is low, most patients’ recovery will have
occurred within 6 months of the injury, although further, slower improvement may
occur in the next 12 to 18 months.4 Therefore, it is ongoing that transplanting
stem cells to the cerebral lesion area and inducing them to differentiate to
neurons substitute neuronal function.
Stem cells are classically defined as cells that have the ability to renew
themselves continuously and possess multipotent ability to differentiate into
many cell types.5 Human bone marrow mesenchymal stem cells (BMSCs) have been
widely studied because of their relative easy access and differentiation
potential to the osteogenic, adipogenic, chondrogenic lineages, hepatocytes,
cardiomyocytes, neurons, and other kinds of tissues or cells.6 Their multipotentiality and self-renewal have increased the attention to this stem
cell model as a self-renewing cell source, with applications in tissue
engineering and regenerative medicine.7 For example, the osteogenic potential of
BMSCs has been explored extensively in the biological evaluation of bone tissue
engineering scaffolding structures.8, 9 Furthermore, several studies have
reported that transplanted BMSCs accelerate neuroplasticity and facilitate
neuronal regeneration, as well as functional recovery.10-14
Therefore, BMSC therapy may be a novel method to repair brain lesions and
promote patients with functional disorders after severe TBI. Here, we report our
experiences with autologous BMSC transplant that we have used in a clinical
trial for patients with TBI complications.
Materials and Methods
Introduction of patients
The study was approved by and registered by the ethical committee of the
Hospital and Health Bureau of City and patients gave their informed consent. The
research reported in the paper was undertaken in compliance with the 1975
Helsinki Declaration and the International Principles. Forty-five patients who
presented with a vegetative state and 121 patients who showed disturbance in
their motor activity after severe TBI for at least 1 month were admitted to the
department of neurosurgery. These patients had a diagnosis of severe TBI based
on clinical evidence and neuroimaging, most prominently a radiologic test, for
example computed tomography and magnetic resonance imaging. And these patients
did not have other serious complications (eg, cachexia, pulmonary infection, and
gastrointestinal bleeding). Ninety-seven patients (24 patients with vegetative
state and 73 patients with disturbance motor activities) received BMSC
transplant voluntarily. All the patients in the trial had been stabilized before
the cell therapy with no apparent improvements in their motor activities and
consciousness. Before regular investigations and therapy, the blood routines and
serum biochemical indexes of these patients were checked to exclude
inflammation, liver and renal insufficiency, and blood diseases. The study is a
nonrandom, open-labeled, interventional cohort study.
Bone marrow mesenchymal stem cell recovery
The biological material used in this study would have been otherwise discarded
during a standard surgical procedure. The procedure of isolating BMSCs refer to
the steps reported in the studies.15, 16 About 100 mL of bone marrow was
recovered by multiple aspirations in the posterior iliac crests in a heparinized
(1 mL/5000 U) bottle and diluted in Dulbecco’s phosphate buffered saline
(without calcium and magnesium) at a ratio of 1:2. This was performed under
sterile conditions with local anesthesia in the operating suite. The obtained
solution was collected and filtered with a 70-µm cell strainer (Falcon,
Pittsburgh, PA, USA) before centrifugation at 400 g for 10 minutes. The cell
interface was carefully removed, and washed twice in Dulbecco’s phosphate
buffered saline at 400 g for 10 minutes. The resultant pellet was added with red
blood cell lysing solution (0.7% ammonium chloride) and incubated for 2 minutes.
Lysing was arrested by adding 0.9% ice-cold sodium chloride, and the cells were
washed in Dulbecco’s phosphate buffered saline until the lysing factors were
removed. Cell pellets were resuspended in Dulbecco’s modified Eagle’s Medium
St. Louis, MO, USA), supplemented with 10% Fetal Bovine Serum (Invitrogen
Corporation, Carlsbad, CA, USA) and 1% antibiotics (streptomycin and penicillin)
(Invitrogen), and cultured in 25-cm2 flasks at 37°C in a humidified atmosphere
containing 5% CO2. On day 4, the cultures were washed with phosphate buffered
saline to remove the nonadherent cells. Finally, cell pellets were resuspended
in 5 mL (about 1 × 106 cells/mL).
Identification of bone marrow stem cells
The International Society for Cellular Therapy proposed a set of minimal
criteria for the characterization of BMSCs, which includes the capability of
adherence to plastic surfaces and the expression of the cell surface markers
CD73, CD90, and CD105.17 In this study, BMSCs were identified by examining the
markers above as the fluorescence-activated cell sorting characterization of the
stem cells. About 100-µL cell samples were incubated with CD73, CD90, and CD105
antibodies conjugated with PE and FITC (BD Biosciences, San Jose, CA, USA)
at a concentration of 2 µg/mL for 15 minutes at
room temperature in the dark. After that, 1 mL
of phosphate buffered saline was added to the stained cells and mixed well.
Then, 5 µL of the
7-aminoactinomycin D dye was added and again incubated in the dark for 10
minutes at room temperature. The cells were analyzed by flow cytometry.
Installation of bone marrow stem cells
Ninety-seven patients decided to receive stem cell therapy by lumbar puncture.
First, we made sure that the localized bacterial infection of the pars lumbalis
skin did not exist before lumbar puncture. The patients had been subjected to
local anesthesia. The BMSCs suspension (5 mL) was installed into
the subarachnoid space by lumbar puncture between the lumbar vertebrae L3/L4 or
Cell suspension was slowly injected into the subarachnoid space for 2 to 3
minutes. Then, patients were maintained in a supine position for 24 to 48 hours.
Fourteen days after therapy, the patients were followed-up for scheduled
Persistent vegetative state evaluation
The vegetative state is a clinical condition of complete unawareness of the self
and the environment accompanied by sleep-wake cycles with either complete or
partial preservation of hypothalamic and brainstem autonomic functions. The
vegetative state can be diagnosed according to the criteria described in the
book.18 The persistent vegetative state (PVS) can be defined as a vegetative
state present at 1 month after acute traumatic or nontraumatic brain injury, and
present for at least 1 month in degenerative/metabolic disorders or
developmental malformations. In this study, improvements of PVS patients were
evaluated according to the grade principle of PVS (drafted by Chinese Medical
Association in Nanjing, China, 1996).
Motor function evaluation
Most motor disorders lack a distinctive biomarker, so evaluation of motor
disorders is primarily clinical, based on careful neurologic examination. The
clinical features of traumatic motor disorders include: (1) increased tone of
(2) weakness most evident in antigravity muscles, (3) increased reflexes and
clonus, (4) shocklike contractions of muscles, and (5) uncoordinated muscle
The chi-square, Fisher exact test, and 1-way analysis of variance were performed
to analyze the data. All tests were considered significant at P values that were
less than .05. Statistical analyses were performed with SPSS software (SPSS: An
IBM Company, version 12.0, IBM Corporation, Armonk, New York, USA).
Safety of cell therapy
Although 5 patients expressed transient fever, and
2 patients felt light headache on second day after the cellular therapy, no
patient experienced any serious adverse event (such as inflammation, systemic
infections, and gastrointestinal bleeding) upon BMSCs reinfusion. No
complications or wound infections were observed in the patients after the
Results of cellular therapy
Fourteen days after cellular therapy, 38 of 97 patients (39.2%) with TBI
improved (P = .007) (Table 1). Eleven patients begin to considerably show posttherapeutic improvements in consciousness
(P = .024) (Table 2); they expressed responsive eyeball tracking occasionally,
groaning, or tearing. Twenty-seven of 73 patients (37.0%) showed improvements in
motor functions (P = .025) (Table 3). The patients with hemiplegic paralysis
showed motor power and scale enhanced after the cellular therapy. The patients
with muscle spasticity expressed muscular tension relaxed partly. The age of
patients influenced the outcome of the cellular therapy (P < .05) (Tables 4 and
5), and the time elapsed between injury and therapy had roles on the outcome of
the therapy on motor disorder (P < .05) (Tables 4 and 5). No correlation was
found between the number of cell injections and improvement
(P > .05) (Tables 4 and 5).
All severity levels of TBI have the potential to cause significant, long-lasting
disability.19 Permanent disability is thought to occur in 100% of severe
injuries.20 Prognosis worsens with the severity and location of the lesion and
depends on the access to immediate, effective acute management.21 It is
important to begin emergency treatment within the acute stage. In the subacute
stage, prognosis is strongly affected by the patient's involvement in activity
that promotes recovery.1, 2 The results of TBI vary widely in type and duration;
they include physical, cognitive, emotional, and behavioral complications.
Traumatic brain injury can cause prolonged or permanent influences on
consciousness, for example PVS. Movement disorders that may develop after TBI
include tremor, ataxia (uncoordinated muscle movements), myoclonus (shocklike
contractions of muscles), and loss of movement range and control (in particular
with a loss of movement repertoire).22
Loss of cellular components and myelination that occur as an inflammatory
process hamper functional recovery.15 Therefore, reducing progressive tissue
damage and scarring, facilitation of remyelination, and re-establishment of lost
neural tissue and its circuitry should be addressed for any successful cellular
therapy. Bone marrow stem cells are multipotent adult progenitor cells that can
differentiate into a variety of cell lineages,23 which make BMSCs excellent
candidates as therapeutic cells for the repair of damaged tissue. In the case of
severe tissue damage, BMSCs can be attracted to the damaged sites.24 The
migration of BMSCs to injury sites, where they secrete bioactive factors that
trophically influence the repair and regenerative process, produce factors that
inhibit scarring and apoptosis, promote angiogenesis and stimulate host
progenitors to divide, and differentiate into neurons and astrocytes to repair
the injured tissue, leading to improved function.25, 26 Further, magnetic
resonance imaging volumetric data reveal no significant change in grey matter,
white matter, intracranial volume, or cerebral spinal fluid space at 1 and
6 months as measured related to expected norms.27 In this regard, the positive
effects of BMSCs may have important clinical use.
In this study, we isolated BMSCs from the bone marrow of the patients themselves
and transplanted stem cells back into the patients. Excitingly, 14 days after
BMSC therapy, 38 of 97 patients who received the stem cell therapy had
improvements. The mechanisms of potential therapeutic benefit of BMSC therapy
may be as follows28: the secretion of growth factors, the exchange of genes and
proteins through cell-to-cell fusion or contact, the induction of angiogenesis,
and the effects on immune modulation. However, there have been numerous
conflicting reports regarding stem cell engraftment and therapeutic efficiency.
In this study, 59 of 97 patients who received the stem cell therapy did not
improve. The reasons might be related to the effects of media, cell passage
number/techniques, or isolation methods.28 The authors also found that BMSC
therapy had more-definite effects on younger patients. The probable reason was
that young patients were in better body basal condition or bone marrow condition
than older ones. Our results revealed an inverse relation between the time
elapsed after the injury and the outcome of cellular therapy. Maybe 1 reason for
this is that scar tissue forms in the damaged site after a longer elapsed time
after the injury and stops migration of stem cells.
Additionally, increasing amounts of research are being conducted to evaluate
multiple routes for delivery of stem cells, such as intravenous, intra-arterial,
and direct routes. Intravenous administration offers easy access to the
circulation, with the possibility of distribution throughout multiple tissues.29
An initial drawback of intravenous application is the large proportion of
first-pass pulmonary sequestration.30 Intra-arterial administration offers a
method to further localize the placement of stem cells. However, recent
investigation has shown that intracarotid infusion of MSCs induces ischemic
stroke.31 Direct or intracerebral implantation of stem cells would maximize the
stem cell load at the site of injury.32 But investigators must consider the
invasiveness of the intracerebral approach and the possibility of further tissue
damage during cell transplant. Lumbar puncture delivery of BMSCs appears to be
superior to other methods. Cell engraftment and tissue sparing were
significantly better after lumbar puncture delivery, and host immune response
after lumbar puncture delivery was reduced.33 When BMSCs were introduced through
a lumbar puncture, stem cells prevented astrogliosis and microglial activation,
and spared and regenerated motoneurons.34 In this study, we performed BMSCs
transplant by lumbar puncture and no complications appeared.
This study suggests that BMSC therapy is safe and effective on patients with
severe TBI complications, such as PVS and motor disorder, through lumbar
puncture. Young patients improve more than older ones do. The earlier the
cellular therapy begins in the subacute stage of traumatic brain injury, the
better the results.
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- Pavlichenko N, Sokolova I, Vijde S, et al. Mesenchymal stem cells
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- Andrews EM, Tsai SY, Johnson SC, et al. Human adult bone marrow-derived
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Volume : 11
Issue : 2
Pages : 176-181
From the 1Institute of Neurology, the First College of Clinical Medical Science,
China Three Gorges University; and the 2Department of Neurosurgery, Yichang
Center People’s Hospital, Yichang, China.
Corresponding author: Xiongwei Wang MD, PhD, Institute of Neurology, the First
College of Clinical Medical Science, China Three Gorges University; Department
of Neurosurgery, Yichang Center People’s Hospital, Yi-Ling-Da-Dao, 183, Yichang
443003, Hubei, People’s
Phone: +86 0717 648 6087
Mobile: +86 1390 860 0067
Fax: +86 0717 648 6087
Table 1. The Results of Cell Therapy on TBI (Case)
Table 2. The Improvements on Posttraumatic PVS After Cell Therapy (Case)
Table 3. Improvements on Motor Disorder After Cell Therapy (Case)
Table 4. PVS After TBI: Group Assessment
Table 5. Motor Disorder After TBI: Group Assessment