Human Clinical Trial - Bone Marrow Stem Cells and Polymer Hydrogels

[litespeed4]

I haven't been posting for a while since I been a little busy with work and research. I contacted this researcher last month about her work and was sent the following paper. If you read past the "rats walking" part you will see the results of a phase 1 clinical trial involving acute and chronic patients.
I'll leave the rest for you read for yourself.

[challenged athlete]

Originally Posted by litespeed4

I haven't been posting for a while since I been a little busy with work and research. I contacted this researcher last month about her work and was sent the following paper. If you read past the "rats walking" part you will see the results of a phase 1 clinical trial involving acute and chronic patients.
I'll leave the rest for you read for yourself.

hey speed, that link is not working.............

[litespeed4]

It seems to work for me. Do you have adobe acrobat installed?
Is anyone else have a problem with this link?

[artsyguy1954]

Originally Posted by litespeed4

It seems to work for me. Do you have adobe acrobat installed?
Is anyone else have a problem with this link?

No, I don't. But I have Adobe Reader 7.0 installed. This Czech researcher seems to do very good work.

[Wise Young]

litespeed,

Thanks for posting this. I have known Eva Sykova for a long time (30 years! since I did my PhD thesis on spreading depression in 1975 and she worked on spreading depression then also). She is a very good researcher.

I find it very interesting that intra-arterial injection of the bone marrow cells is better than intravenous injection, consistent with a possible beneficial effect of the drug. Please note that most of the patients were "subacute" which means that most of them should be some improvement in both motor and sensory ASIA scores. The treatment appears to be safe.

Wise.

P.S. for those who are downloading the paper, it is about 0.5 Mb and takes about a minute or so. People have to be patient and the link does work.

[Duran]

Information removed at request of member.

[Susan Fajt]

Thought i would question whom out of *all* of these "therapies" are best and how do we accomplish getting past the rat/primate studies or have some of them already regarding the "refrences" and what are the outcomes as i find this to be very intriguing as to where are these researchers now with human clinical trials? It is very obvious that ALOT of RESEARCH FOR THE SAKE OF RESEARCH is going on and imo a cure has been found and many years ago i might add.. hmmmmm



Cellular and Molecular Neurobiology ( C
2006)
DOI: 10.1007/s10571-006-9007-2
Bone Marrow Stem Cells and Polymer Hydrogels—Two
Strategies for Spinal Cord Injury Repair
Eva Sykov´ a,1,2,3 Pavla Jendelov´ a,1,2 Lucia Urdz´ıkov´ a,1,2 Petr Lesn´ y,1,2
and Aleˇs Hejˇcl1,2
Received November 02, 2005; accepted January 05, 2006
SUMMARY
1. Emerging clinical studies of treating brain and spinal cord injury (SCI) led us to
examine the effect of autologous adult stem cell transplantation as well as the use of
polymer scaffolds in spinal cord regeneration. We compared an intravenous injection of
mesenchymal stem cells (MSCs) or the injection of a freshly preparedmononuclear fraction
of bone marrow cells (BMCs) on the treatment of an acute or chronic balloon-induced
spinal cord compression lesion in rats. Based on our experimental studies, autologous BMC
implantation has been used in a Phase I/II clinical trial in patients (n=20) with a transversal
spinal cord lesion.
2. MSCs were isolated from rat bone marrow by their adherence to plastic, labeled
with iron-oxide nanoparticles and expanded in vitro. Macroporous hydrogels based on
derivatives of 2-hydroxyethyl methacrylate (HEMA) or 2-hydroxypropyl methacrylamide
(HPMA) were prepared, then modified by their copolymerization with a hydrolytically
degradable crosslinker, N,O-dimethacryloylhydroxylamine, or by different surface electric
charges. Hydrogels or hydrogels seeded with MSCs were implanted into rats with hemisected
spinal cords.
3. Lesioned animals grafted with MSCs or BMCs had smaller lesions 35 days postgrafting
and higher scores in BBB testing than did control animals and also showed a
faster recovery of sensitivity in their hind limbs using the plantar test. The functional improvement
was more pronounced in MSC-treated rats. InMR images, the lesion populated
by grafted cells appeared as a dark hypointense area and was considerably smaller than
in control animals. Morphometric measurements showed an increase in the volume of
spared white matter in cell-treated animals. In the clinical trial, we compared intraarterial
(via a. vertebralis, n=6) versus intravenous administration of BMCs (n=14) in a group
of subacute (10–33 days post-SCI, n=8) and chronic patients (2–18 months, n=12). For
patient follow-up we used MEP, SEP, MRI, and the ASIA score. Our clinical study revealed
that the implantation of BMCs into patients is safe, as there were no complications
following cell administration. Partial improvement in the ASIA score and partial recovery
of MEP or SEP have been observed in all subacute patients who received cells via
a. vertebralis (n=4) and in one out of four subacute patients who received cells intravenously.
Improvement was also found in one chronic patient who received cells via a.
vertebralis. A much larger population of patients is needed before any conclusions can be
drawn. The implantation of hydrogels into hemisected rat spinal cords showed that cellular
ingrowth was most pronounced in copolymers of HEMA with a positive surface electric
1 Institute of Experimental Medicine ASCR, Prague, Czech Republic.
2 Center for Cell Therapy and Tissue Repair and Department of Neuroscience, Second Medical Faculty,
Charles University, Prague, Czech Republic.
3To whom correspondence should be addressed at Institute of Experimental Medicine ASCR, Vidensk´a
1083 142 20 Prague 4, Czech Republic; e-mail: sykova@biomed.cas.cz.
0272-4340/06 C
2006 Springer Science+Business Media, Inc.
Sykov´ a, Jendelov´ a, Urdz´ıkov´ a, Lesn´ y, and Hejˇcl
charge. Although most of the cells had the morphological properties of connective tissue
elements, we found NF-160-positive axons invading all the implanted hydrogels from both
the proximal and distal stumps. The biodegradable hydrogels degraded from the border
that was in direct contact with the spinal cord tissue. They were resorbed by macrophages
and replaced by newly formed tissue containing connective tissue elements, blood vessels,
GFAP-positive astrocytic processes, and NF-160-positive neurofilaments. Additionally, we
implanted hydrogels seeded with nanoparticle-labeled MSCs into hemisected rat spinal
cords. Hydrogels seeded with MSCs were visible on MR images as hypointense areas, and
subsequent Prussian blue histological staining confirmed positively stained cells within the
hydrogels.
4. We conclude that treatment with different bone marrow cell populations had a
positive effect on behavioral outcome and histopathological assessment after SCI in rats;
this positive effect was most pronounced following MSC treatment. Our clinical study
suggests a possible positive effect in patients with SCI. Bridging the lesion cavity can
be an approach for further improving regeneration. Our preclinical studies showed that
macroporous polymer hydrogels based on derivatives of HEMA or HPMA are suitable
materials for bridging cavities after SCI; their chemical and physical properties can be
modified to a specific use, and 3D implants seeded with different cell types may facilitate
the ingrowth of axons.
KEY WORDS: cell transplantation; magnetic resonance; spinal cord lesion; repair;
scaffold; polymers.
STRATEGIES FOR SPINAL CORD INJURY TREATMENT
Spinal cord injury (SCI) invariably results in the loss of neurons and axonal degeneration
at the lesion site, leading to severe functional impairment, paraplegia, or
tetraplegia. Currently, there is no effective treatment for SCI. Standard treatment
consists of stabilization and a conservative therapy using high doses of methylprednisolone
(Hugenholtz et al., 2002), although there have been clinical studies with
naloxone (Bracken et al., 1990), tirilazade (Bracken et al., 1998), GM-1 ganglioside
(Geisler et al., 2001),TRH(Pitts et al., 1995), and nimodipine (Pointillart et al., 2000).
Long-term therapy after spinal cord injury focuses on rehabilitation, pain relief, spasticity
treatment, and the prevention of complications. Supportive techniques come to
the fore, e.g., functional electrical stimulation, tendon replantation, and neuroprosthetics.
However, the regenerative capacity of the central nervous system (CNS) is
highly limited and, in the region of a trauma, a glial scar develops rather than normal
tissue (Fawcett and Asher, 1999). The scar contains various substances inhibiting
axonal growth and forms a mechanical barrier that separates the injured tissue from
the rest of the CNS. The restoration of tissue function in the injured region requires
the development of methods that allow for the reconstruction of grey and white
matter composed of elements of central nervous system tissue (neurons, astrocytes,
oligodendrocytes, blood vessels, extracellular matrix components, and myelinated
nerve fibres).
The development of spinal cord injury treatment is focused on new methods of
regenerative medicine such as the administration of growth factors, e.g., PDGF and
NT3, or antagonists of inhibiting molecules (NOGO), preventing the deposition of
extracellularmatrixmolecules (chondroitin sulphate), and stimulating the growth of
damaged axons as well as protecting them from further damage (Bixby and Harris,
1991; Venstrom and Reichardt, 1993; Lee et al., 2003). An emerging strategy for
Therapy for Spinal Cord Injury
replacing and/or regenerating damaged tissue is the implantation of stem cells and/or
artificial biomaterials such as scaffolds to form tissue bridges between damaged spinal
cord stumps. Recent preclinical studies include the implantation of foetal nervous
tissue (Bregman, 1987), embryonic stem cells (Brustle et al., 1999), bone marrow
mesenchymal stem cells (Prockop, 1997;Akiyama et al., 2002a; Jendelov´a et al., 2003,
2004b), Schwann cells (Kuhlengel et al., 1990), peripheral nervous tissue (Wrathall
et al., 1982; Horvat, 1991), collagen-based matrices containing cells or neuroactive
substances (Houweling et al., 1998; Liuet al., 1998), nitrocellulose membranes (Houle
and Ziegler, 1994), tubes made from polymeric materials (Houle and Ziegler, 1994;
Xu et al., 1997; Oudega et al., 2001), polymer hydrogels (Woerly et al., 1998, 2001a,b;
Lesn´y et al., 2002; Jendelov´a et al., 2004a), and biodegradable polylactide implants
(Maquet et al., 2001).
TRACKING OF IMPLANTED CELLS IN VIVO
Cell transplantation represents a potentially powerful treatment method for
spinal cord injury; however, current experiments with stem cells do not give us
information about the behavior of the transplanted cells in the host organism
in vivo, especially about their migration and fate within the target structures and
their potential neoplastic growth. The lack of these data can create a serious obstacle
for the therapeutic use of cell therapy. Human medicine would benefit from
the labeling of implanted stem cells and the use of noninvasive methods to image
the labeled cells postimplantation. It has been shown that MSCs or ESCs can
be labelled with superparamagnetic iron-oxide nanoparticles. Nanoparticle-labeled
cells, when implanted into rats with a cortical or spinal cord lesion, are visible
on MR images as a sharply bounded hypointense area at the injection site, and
subsequently a hypointense signal is also seen at the lesion site. The cells can
migrate along the corpus callosum and, if injected intravenously, can cross the
BBB and home to a cortical or spinal cord lesion. The hypointense signal on MR
images persists in brain or spinal cord for more than 50 days (Jendelov´a et al.,
2003, 2004b; Sykov´a and Jendelov´ a, 2005). Nanoparticles, based on dextran-coated
iron oxide, e.g., Endorem (Guerbet, France), can therefore be used as a stem cell
marker for noninvasive MR tracking of cell migration and fate following transplantation.
The use of MRI to monitor transplanted cells will be directly applicable
to human medicine to monitor the course, results and potential risks of cell
therapy.
BONE MARROW CELLS AS A TOOL FOR SPINAL CORD
INJURY REPAIR
The use of bone marrow stromal cells (MSCs) in cell therapies may have some
advantages over the use of other sources of cells: they are relatively easy to isolate
from bone marrow, they grow well in tissue cultures, they may be used in autologous
transplantation protocols and bone marrow as a source of cells has been already
Sykov´ a, Jendelov´ a, Urdz´ıkov´ a, Lesn´ y, and Hejˇcl
approved for the treatment of hematopoietic diseases. Preclinical studies have been
performed on rats with a spinal cord injury and have shown that transplanted cells
in the injured spinal cord survive, migrate into the host tissue and lead to axonal regeneration
and motor function recovery (Sasaki et al., 2001; Akiyama et al., 2002a,b;
Hofstetter et al., 2002; Chopp and Li, 2002; Inoue et al., 2003; Jendelov´a et al., 2004b).
This has been achieved not only through the implantation of bone marrow stromal
cells (MSCs), but also through the implantation of freshly collected bone marrow
containing nucleated cells (BMCs) with a relatively small percentage of mesenchymal
cells (Sasaki et al., 2001; Akiyama et al., 2002b; Inoue et al., 2003; Sykov´a et al.,
2005b; Urdz´ıkov´a et al., in press). Two clinical studies so far have shown the safety
of such an approach and the partial improvement of function in acute patients (Park
et al., 2005; Sykov´a et al., 2005b).
We have shown that the intravenous injection of MSCs, BMCs, or G-CSF significantly
improves the recovery of hindlimb motor function in rats with a spinal
cord compression lesion (Sykov´a et al., 2005a; Urdz´ıkov´a et al., in press). The fate
of transplanted MSCs labeled with iron-oxide nanoparticles was followed by ex vivo
MRI (Sykov´a and Jendelov´ a, 2005). Locomotor function was assessed weekly for
5 weeks after SCI by the BBB test (Basso et al., 1996); hindlimb sensitivity was
tested over the same time period using the plantar test. Lesioned animals grafted
with MSCs or BMCs had higher scores in BBB testing than did control animals
injected with saline (Fig. 1(A)). All cell-treated animals showed a recovery of hind
limb sensitivity using the plantar test; recovery was most apparent and most rapid
in animals grafted with MSCs (Fig. 1(B)). Similar results were seen after the injection
of G-CSF. Locomotor function and hindlimb sensitivity significantly improved
35 days after treatment. MR images of transversal and longitudinal spinal cord sections
from animals grafted with magnetically labeled MSCs showed the entire lesion
as a dark hypointense area (Fig. 2(A and B)). Staining for iron (Prussian blue) revealed
many cells labelled with nanoparticles in the lesion site, and the lesion cavities
were significantly smaller than in control animals. (Fig. 2(C and D)). Morphometric
measurements of the spared white and grey matter were performed in the centre
of the lesions. The spared cross-sectional area of the white matter, as well as the
volume of spared white matter, was significantly greater in all cell-treated animals.
The spared cross-sectional area of the grey matter was significantly larger only in
MSC treated animals, while the volume of spared grey matter was not significantly
influenced by cell treatment (Fig. 2(E)).
Based on these studies in which the cells were implanted 1 week after spinal
cord injury, we studied the effect of an intravenous injection of MSCs on functional
outcome in chronically injured rats (Urdz´ıkov´a et al., 2005). The rats underwent
a spinal cord compression lesion and 4 months later were intravenously injected
with MSCs. Rats were tested behaviourally using the locomotor open field BBB
test, and the plantar test was used to assess hindlimb sensitivity. Morphometric
analysis was performed to evaluate the extent of spared white and grey matter
in 11 mm long lesion-centered spinal cord segments. The volume of the spared
tissue in these 11 mm long segments was calculated as the sum of cross-sectional
areas multiplied by the distance between them. Our results demonstrate that the
intravenous transplantation of MSCs 4 months after spinal cord compression injury
Therapy for Spinal Cord Injury
Fig. 1. Behavioural testing after treatment with MSCs
and BMCs. A: Behavioural open field BBB motor scores
of MSC- and BMC-treated rats were significantly higher
than those of control animals 14, 21, 28, and 35 days after
SCI (p < 0.05). B: Time course of the animals’ response
to radiant heat measured by the plantar test in treated and
control rats. In all treated rats, latency times decreased
as their recovery progressed. The most prominent effect
was seen in MSC-treated rats. The latency time in salineinjected
(control) rats did not change during the 35-day
survival period. Data are averaged between right and left
hind limbs and expressed as mean ± SEM. ∗p < 0.05
compared to control group.
had no effect on motor function expressed as the BBB score but it significantly
improved sensitivity as measured by the plantar test. The total volume of the spared
grey and white matter in the lesion epicentre was not significantly different between
MSC-treated animals and controls, but morphometric analysis showed a significant
increase in the cross-sectional area of spared white and grey matter caudally to the
lesion centre. The effect of MSCs implanted into rats with SCI is therefore more
pronounced when the cells are grafted 7 days after lesioning rather than 4 months
postinjury.
Sykov´ a, Jendelov´ a, Urdz´ıkov´ a, Lesn´ y, and Hejˇcl
Fig. 2.
Therapy for Spinal Cord Injury
HYDROGELS AND CELL–POLYMER CONSTRUCTS
The importance of biomaterials has steadily increased in recent years, and the
number of polymer applications in tissue engineering continues to grow. Since CNS
injury (particularly spinal cord injury) is accompanied by cell death, pseudocyst
formation and glial scarring, which compromise regeneration in the injured region,
extensive research is currently focused on the development of treatment approaches
that prevent scarring and bridge the lesion cavities. Macroporous biocompatible
hydrogels can be used to eliminate scarring, bridge cavities and facilitate regeneration
(Woerly et al., 1998, 2001b). These hydrogels are highly biocompatible, and when
implanted into nervous tissue they are known to be chemically inert and nontoxic
(Pˇr ´adn´y et al., 2002). They have a high water content (70–90%) and a very large
surface area, and they are macroporous with pore sizes of 10–50 µm (Fig. 3(A))
(Lesn´y et al., 2002; Pˇr ´adn´y et al., 2002).
Spinal cord tissue regeneration was studied following the implantation of polymer
hydrogels with a macroporous structure, based on derivatives of 2-hydroxyethyl
methacrylate (HEMA), 2-hydroxypropyl methacrylamide (HPMA) or copolymers
of HPMA and ethoxyethyl methacrilate (EOEMA) (Woerly et al., 1998; Pˇr ´adn´y
et al., 2002, in press). The hydrogels were modified either by different surface
electric charges (HEMA-MA negative charge; HEMA-MOETACl positive charge)
or by their copolymerization with a hydrolytically degradable crosslinker, N,Odimethacryloylhydroxylamine
(Pˇr ´adn´y et al., in press). Blocks of hydrogel were
implanted into hemisected rat spinal cords; the animals were sacrificed 28 days after
implantation. All of the hydrogels were biocompatible and adhered well to the
host tissue, bridging the whole spinal cord lesion (Fig. 3(B)); cellular ingrowth was
observed in all the implanted hydrogels, with the most pronounced ingrowth seen in
copolymers ofHEMAwith a positive electric charge.Although most of the cells had
the morphological properties of connective tissue elements, NF-160-positive axons
were found invading all the implanted hydrogels from both the proximal and distal
stumps. No astrocytic processes inside the gels were observed; however, the gels
were permissive for Schwann cells (p75-positive) (Jendelov´a et al., 2004a).
The biodegradable hydrogels degraded from the borders that were in direct
contact with the spinal cord tissue (Fig. 3(C)). They were resorbed by
macrophages and replaced by newly formed tissue containing connective tissue elements,
blood vessels, GFAP-positive astrocytic processes, and NF-160-positive neurofilaments
(Fig. 3(D and E)). The size of the degraded zone was dependent on the
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Fig. 2. MSCs labeled with nanoparticles implanted into rats with a spinal cord compression lesion.
A,B: Transversal and longitudinal images of a spinal cord compression lesion populated by intravenously
injected nanoparticle-labeled MSCs, four weeks after implantation. The lesion with nanoparticle-labeled
cells is visible as a dark hypointensive area. C: Prussian blue staining of a spinal cord compression lesion
(control animal).D: Prussian blue staining of a spinal cord lesionwith intravenously injected nanoparticlelabeledMSCs.
Note the smaller lesion size in the implanted animal than in the control. Insert: The lesion
is populated with Pussian blue-positive cells. A–D Modified from (Jendelov´a et al., 2004b). E: Analysis of
the spared white and grey matter volume in the centre of the lesion. The total volume of the white matter
(WM) and grey matter (GM) in 11 mm long segments of the spinal lesion in treated and saline injected
animals. No statistically significant differences in spared grey matter volume were observed between the
groups. Data are represented as group means ± SEM. ∗p < 0.05 compared to control group.
Sykov´ a, Jendelov´ a, Urdz´ıkov´ a, Lesn´ y, and Hejˇcl
Fig. 3.
Therapy for Spinal Cord Injury
degradation rate of the hydrogel (Pˇr ´adn´y et al., in press); The largest degraded zone
was found in hydrogels biodegradable within 7 days, comprising approximately onehalf
of the implant volume. The central part of the hydrogels consisted of amorphous
matter, where only the ingrowth of connective tissuewas observed. This is most likely
the result of the hindered clearance of degradation products from the centre of the
gel (Pˇr ´adn´y et al., in press).
The formation of lesion cavities in chronic injuries is one of the factors inhibiting
neuronal regeneration. HEMA hydrogels were tested for their ability to
bridge lesions in rats with a complete spinal cord transection at the Th9 level (Hejˇcl
et al., 2005). The lesions were bridged using hydrogels implanted either immediately
or after a one week delay. Histological assessment was performed 3 months after
transection. Histological staining showed that the hydrogels were filled with connective
tissue elements, blood vessels, and neurofilaments (Fig. 3(H)). Morphometric
analysis of the spinal cord transection lesions showed that the volume of the pseudocysts
formed in the spinal cords with delayed hydrogel implantation was significantly
smaller than the volume of the cavities in rats treated by immediate hydrogel implantation
(Fig. 3(F and G)). The results indicate that delayed implantation may be
even more effective than immediate reconstructive surgery.
Hydrogel implantation can be combined with stem cell grafting: Before transplantation
into a lesion, the hydrogels are seeded with MSCs. In this case the hydrogels
form an inert environment, allowing for the free diffusion of intrinsic growth
factors, in which the cells start to differentiate and migrate. The inert environment of
the hydrogels also provides an adequate standard background forMRimaging of the
cells (Jendelov´a et al., 2004a).We employed cell–polymer constructs in order to facilitate
the regeneration of injured spinal cord (Lesn´y et al., in press). In macroporous
polymer hydrogels based on HEMA with an average porosity of 50 µm, the cellular
growth was distinctly influenced by the surface electric charge. In negatively charged
hydrogels (HEMA-MA), the cells grew in clusters uniformly scattered within the
hydrogel volume; these clusters had minimal contact with the hydrogel surface. In
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Fig. 3. The use of hydrogels in spinal cord injury repair. A: The structure of aHEMAhydrogel observed
using a scanning electron microscope. B: Four weeks after implantation, aHEMAhydrogel firmly adheres
to the spinal cord tissue. The dotted line marks the interface between spinal cord tissue and the hydrogel,
which is fully integrated into the spinal cord. C: A biodegragable hydrogel (HPMA +10% EOEMA)
degraded from the interface with the spinal cord towards the central part of the hydrogel. The size
(volume) of the peripheral zone of degraded hydrogel is marked by the black line. The central part
of the hydrogel (marked with an asterisk) consisted of amorphous matter, where mostly the ingrowth
of connective tissue and capillaries was observed. D: The detail of the peripheral zone of a degraded
hydrogel. The gel was resorbed by macrophages and replaced by newly formed tissue. E: The detail of
the peripheral zone of a degraded hydrogel. NF160-positive neurofilaments are entering the gel from the
spinal cord border. F: Interface between the spinal cord stump and a hydrogel acutely implanted into a
complete spinal cord transection. G: Interface between the spinal cord stump and a hydrogel implanted
seven days after complete spinal cord transection. Note the smaller cavity with delayed implantation. H:
Immunostaining with anti-neurofilament antibody (anti-NF160) showing the presence of NF160-positive
axons in the centre of a hydrogel inserted 7days after complete spinal cord transection. Images C–H
were taken four weeks postimplantation. I: Endorem-labeled cells seeded into a hydrogel. J: A hydrogel
seeded with MSCs was visible on MR images 6 weeks after implantation as a hypointensive area (arrow).
K: Ingrowth of NF 160-positive neurofilaments into a hydrogel seeded with MSCs. I–J Modified from
(Sykov´a and Jendelov´ a, 2005).
Sykov´ a, Jendelov´ a, Urdz´ıkov´ a, Lesn´ y, and Hejˇcl
hydrogels with a positive surface charge (HEMA-MOETACl), the cells adhered well
to the hydrogel surfaces and grew to higher culture densities.
To evaluate the ability of cell–polymer constructs to bridge a lesion, the right
half of a spinal cord segment was removed by hemisection and a block of HEMA
hydrogel seeded with Endorem-labeled MSCs was inserted (Fig. 3(I); Sykov´a and
Jendelov´ a, 2005). Six weeks after implantation, the hydrogel had formed a continuous
bridge between the hemisected spinal segments, re-establishing the anatomical
continuity of the tissue. The hydrogel was visible on MRI as a hypointense
area (Fig. 3(J)) and Prussian blue staining confirmed positively stained cells within
the hydrogel. Staining for neurofilaments (NF160 Sigma, St. Louis, USA) showed
axonal ingrowth into the hydrogel (Fig. 3(K); (Jendelov´a et al., 2004a). Hydrogels
seeded with stem cells may therefore serve as an alternative to the conventional
grafting of dissociated cells, benefiting from advances in surface chemistry
and the cell–cell or cell–matrix interactions that occur during development or
regeneration.
ONGOING CLINICAL STUDY
Based on recent experimental studies, autologous BMC implantation is being
used in a Phase I clinical trial in patients (n=20) with a transversal spinal cord lesion
at MotolHospital in Prague, Czech Republic. Ethical approval for this study was obtained
from theMinistry of Health of theCzech Republic and theEthical Committee
of Motol Hospital in Prague. Informed consent was obtained from each patient who
was enrolled in the study; all patients had suffered traumatic SCI and completemotor
and sensory dysfunction. The patients were divided into two groups. The first group
was a group of “subacute” patients (n=8) who received BMCs between 10 and
33 days after SCI. The second group comprised “chronic” patients who received
BMCs between 2 and 18 months after SCI (n=12). In the eight subacute patients,
four received BMCs via catheterization of a. vertebralis and four patients were infused
intravenously. Of the 12 chronic patients, 2 received BMCs via catherization
of a. vertebralis and the rest intravenously. Spinal cord lesions were evaluated using
MRI. At both initial and follow-up examinations (at 3, 6 and 12 months after BMC
transplantation), standard neurological classification of the SCI was performed according
to theAmerican Spinal Injury Association [ASIA] protocol and the Frankel
score, which provides for a standardised assessment of neurological deficits in patients
with SCI. To assess the functional integrity of the corticospinal tract and
dorsal columns, electrophysiological recordings of motor and somatosensory potentials
(MEP and SEP) were performed prior to and at 3, 6, and 12 months after
BMC transplantation. Partial improvement in the ASIA score, along with a partial
recovery of MEP or SEP, has been observed in all subacute patients who received
cells via a. vertebralis (n=4) and in one out of four subacute patients who received
cells intravenously. Improvement was also found in one out of two chronic patients
who received cells via a. vertebralis. The improved ASIA outcome was mostly from
a score of A to B, in one case from B to D. A much larger population of patients is
needed before any conclusions can be drawn (Sykov´a et al., 2005a,b). At present, it
Therapy for Spinal Cord Injury
can be concluded from this clinical study that the implantation of autologous BMCs
is safe, as there were no complications following intravenous or intraarterial cell
administration. We can also conclude that it appears important that implantation is
done during the first 3–4 weeks after injury and that administering the cells closer to
the injury site, such as through the catherization of a. vertebralis, might be justified
by a better outcome.
DISCUSSION AND FURTHER PERSPECTIVES
Postnatal bone marrow has traditionally been seen as an organ composed of
two main systems rooted in distinct lineages: the hematopoietic tissue proper and
the associated supporting stroma—marrow stromal cells.Unlike hematopoietic stem
cells, whose role in the treatment of hematopoietic diseases has been known for a
long time, MSCs were originally examined only because of their critical role in the
formation of the hematopoietic microenvironment.More recent data came with the
recognition that MSCs are stem/progenitor cells of ectodermal, mesodermal and endodermal
tissues. Their potential to differentiate into nonhematopoietic organ cells
granted them membership in the family of somatic stem cells. There is little doubt
that they represent one of the most accessible sources of stem cells for therapeutic
use. The ease with which they are harvested and the simplicity of the procedures
required for their extensive growth in culture, together with easy expansion in vitro,
may make them ideal candidates.
The question of which cell type is most beneficial for SCI treatment is still unresolved.
One possible effect of cell therapy is “repair,” meaning that the grafted
cells integrate into the host tissue and replace damaged or lost cells. Several studies
have been performed using in vitro expanded neural stem/progenitor cells that
were then implanted into injured rat or marmoset spinal cord. The cells survived
and differentiated into neurons, astrocytes and oligodendrocytes and had a positive
effect on functional outcome (Ogawa et al., 2002, Iwanami et al., 2005, Okada
et al., 2005). Similarly, MSCs can also differentiate into neuron-like cells and glia
(Prockop, 1997; Azizi et al., 1998; Eglitis et al., 1999; Brazelton et al., 2000; Mezey
et al., 2000;Woodbury et al., 2000; Jendelov´a et al., 2003). In our previous experiments
(Jendelov´a et al., 2003), we injected MSCs into rats with a cortical photochemical
lesion and studied the differentiation of the grafted cells. We found that only a few
(<5%) BrdU-labeled MSCs expressed the neuronal marker NeuN, and we did not
find any BrdU-labeled MSCs expressing the astrocytic marker GFAP.
To date, preclinical studies have revealed several reasons why MSCs may be
useful in spinal cord injury treatment. A number of studies have described the use
of MSCs as cells that express factors beneficial to the nervous tissue or that activate
compensatory mechanisms and endogenous stem cells within the tissue following
their migration into an injured environment (for review see Chopp and Li, 2002).
Studies of MSCs transplanted into different models of CNS injury (Chopp et al.,
2000; Lu et al., 2001; Akiyama et al., 2002a; Hofstetter et al., 2002; Urdz´ıkov´a et al.,
in press) have provided considerable information about their potential to improve
functional outcome. MSCs secrete cytokines such as colony stimulating factor (CSF),
Sykov´ a, Jendelov´ a, Urdz´ıkov´ a, Lesn´ y, and Hejˇcl
interleukins, stem cell factor (SCF) (Eaves et al., 1991; Majunder et al., 1998), nerve
growth factor (NGF), brain derived neurotrophic factor (BDNF), hepatocyte growth
factor (HGF), and vascular endothelial cell growth factor (VEGF) (Bjorklund and
Lindvall, 2000). It has also been reported that MSCs stimulate glial cells to produce
neurotrophic factors such as nerve growth factor (NGF) and brain-derived
neurotrophic factor (BDNF) (Majunder et al., 1998; Mahmood et al., 2002; Wang
et al., 2002). MSCs can promote axonal regeneration by guiding nerve fibres
(Hofstetter et al., 2002).Wu showed that transplanted MSCs promote compensatory
mechanisms to reorganise neural networks and activate endogenous stem cells (Wu
et al., 2003). It was also shown that BMCs and MSCs improve neurologic deficits by
generating either neural cells or myelin-producing cells (Chopp et al., 2000; Sasaki
et al., 2001). However, understanding the actual differentiation spectrum of stromal
cells requires further investigation.
Although our and other studies indicate that MSCs are more effective in the
treatment of SCI, there are several good reasons supporting the use of BMCs in SCI
therapies. BMCs include hematopoietic stem cells, macrophages, lymphocytes, as
well as marrow stromal cells. One reason is that the identities of the subpopulations
responsible for neuronal differentiation remain unknown. Second, the neuronal
protective roles of not only MSCs, but also of hematopoietic stem cells, are well
known (Chen et al., 2002; Chong et al., 2002). Hematopoietic stem cells secretemany
cytokines, including trombopoietin and interleukin 11 (Mehler et al., 1993; Dame
et al., 2003). These cytokines are also known to be essential factors for the survival
and differentiation of neuronal progenitor cells.
A recent clinical study was performed by Park et al. (2005) on six patients
with SCI. A combination of autologous BMCs implanted as early as 7 days after
SCI and subsequent repetitive mobilization of bone marrow cells with granulocyte
macrophage-colony stimulating factor (GM-CSF) resulted in five out of six patients
showing improved motor and/or sensory function. In our clinical study with BMCs,
we also found partial functional improvement in subacute patients, which corresponds
well to preclinical studies in rats and nonhuman primates (Sasaki et al., 2001;
Akiyama et al., 2002b; Iwanami et al., 2005). Even when we observed an improvement
in the ASIA score, accompanied by enhanced MEP and SEP during electrophysiological
tests, the improvement was generally only from the A to B score and in
one case from B to D. This clinical study shows that the implantation of autologous
BMCs is safe, but we cannot conclude that the observed effects were due to cell
therapy. However, the outcome in one chronic patient, who was in stable condition
for several months prior to cell implantation, is promising. Nevertheless, there seems
to be a similar therapeutic window as in animal studies, which is between 3 days and
3 weeks after SCI. We suggest that administering the cells closer to the injury site,
such as through the catherization of a. vertebralis, might be important for a better
outcome. Clinical studies are necessary for transferring preclinical findings from animal
experiments to humans. The therapeutic window, the implantation strategy, the
method of administration, the number of cells and the possible side-effects can only
be tested in human clinical trials.
However, in the case of large lesions, cells alone are not able to repair the tissue.
It is necessary to fill the gap left by the lost cell population in order to provide support
Therapy for Spinal Cord Injury
for tissue restoration, reduce the glial scar, and create a permissive environment for
cellular ingrowth.Biocompatible polymer hydrogels, based onpHEMA,orpHPMA,
have viscoelastic and adhesive properties that promote their rapid integration at
the host–tissue boundary. Their macromolecular network provides mechanical cues
that stimulate the ingrowth of cells. Water present within the network provides
free space for the diffusion of host tissue extracellular fluids containing trophic
and growth factors released by neighboring cells. Surprisingly, in a model of delayed
tissue restoration (the implantation of hydrogels one week after complete spinal cord
transection), much reduced pseudocyst and cavity formation was observed (Fig. 3(F
and G)). These results may be due to the early removal of myelin debris and lesion
“clearance” by activated microglia and macrophages. Similarly, Bregman’s group
reported the better ingrowth of axons into fetal spinal cord transplants after a delay
of 2–4 weeks following spinal cord transection (Coumans et al., 2001). It is evident
that finding the optimal therapeutic window will play an important role in any type
of SCI treatment.
The chemical and physical properties of hydrogels can be tailored to a specific
use, and the gels can be seeded with different types of stem cells to create cell–
polymer constructs. These constructs may serve as stem cell carriers, delivering the
cells into the lesion cavities and facilitating axonal regeneration. It was shown that
the implantation of scaffold-neural stem cell constructs into an adult rat hemisection
model of SCI (Teng et al., 2002) promoted long-term improvement in function (persistent
for 1 year in some animals) relative to a lesioned control group. At 70 days
postinjury, animals implanted with a scaffold seeded with cells exhibited coordinated,
weight-bearing hindlimb stepping.Histology and immunocytochemical analysis suggested
that this recovery might be attributable partly to a reduction in tissue loss
from secondary injury processes as well as to diminished glial scarring. Tract tracing
demonstrated corticospinal tract fibres passing through the injury epicentre to the
caudal cord, a phenomenon not present in untreated groups. Different types of cell
populations have various properties and regenerative capabilities, therefore more
studies with cell-polymer constructs seeded with different cell populations may be
promising and useful.
Satisfactory outcomes have not been achieved to date in treating SCI by means
of a single approach. Spinal cord injury represents a complex event, and therefore
effective therapeutic strategies will consist of a series of interventions. First,
secondary tissue loss should be prevented through early neuroprotective, antiin-
flammatory, or immunomodulatory interventions. Subsequently, strategies to promote
the regrowth of axons and the restoration of function will involve multiple
approaches: reducing scar formation, overcoming additional inhibitory molecules,
stimulating damaged nerve cells to regenerate axons, facilitating axonal growth
across the site of injury, and enabling the formation of new connections. Our
overview describing the use of bone marrow cells and polymer hydrogels as tools
for SCI repair is only one contribution towards a multifaceted approach to SCI
treatment. The concept of polymer scaffolds seeded with stem cells may provide a
prototype for other multidisciplinary strategies for addressing complex neurological
injuries.
Sykov´ a, Jendelov´ a, Urdz´ıkov´ a, Lesn´ y, and Hejˇcl
ACKNOWLEDGMENTS
Supported byASCR50390512,GACR309/06/1594, 309/06/1246, 1A8697-5/2005,
NR8339-3, and MSMT 1M0021620803, LC554.
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[artsyguy1954]

Originally Posted by Susan Fajt

It is very obvious that ALOT of RESEARCH FOR THE SAKE OF RESEARCH is going on and imo a cure has been found and many years ago i might add.. hmmmmm

I'd like to think you are right about this, Susan, but I am not so sure.

[Susan Fajt]

artsyguy,

I would like to think that i am wrong for once.. The cure is *almost* here after all the research.. lmao. I like to believe that are scientists are smart enough to find and implement a cure and in a more timely manner. Like i said, i believe there is alot of research for the sake of research, hell how could i not as it is obvious to me.?. Unbelievable.

[KIM]

Somewhere I read that researchers are 2 years ahead of what is published.