Interaction
between mesenchymal stem cells and osteogenic cytokines in vitro
and in vivo
Sadanori Akita, MD, PhD, Masashi Fukui, MD, PhD, Tohru Fujii, MD, Kozo
Akino, MD, PhD, Nagasaki University, School of Medicine, Department of Plastic
and Reconstructive Surgery
Cytokines such as bone morphogenetic protein-2 (BMP-2) greatly induces osteoblast
differentiation, while angiogenic cytokines such as basic fibroblast growth
factor (bFGF) can increase the DNA content in vitro. Additionally,
combined treatment with both cytokines showed the synergistic effect in
bone formation in vitro.
First, human mesenchymal stem cells with BMP-2 and bFGF in vitro
were tested with medium containing dexa-methasone, ascorbic acid and b-glyceronphosphate
for cell proliferation and alkaline-phosphatase (ALP) activity.
Then, a nude rat calvarial bone defect model with a gelatin sponge carrier,
mesenchymal stem cells and combined cytokine treatment was used as an orthotopic
in vivo model. A nude rat modified epigastric flap model, which was
newly established by the authors, was used as an ectopic in vivo
model. Normal human mesenchymal stem cells (hMSC), derived from a single
donor, were purchased from Osiris Therapeutics, Inc. (Cat # CCS-PT-2501).
The cells were initially cryopreserved and thawed in medium containing L-Glutamine,
Penicillin and Streptomycin. The medium was exchanged every three days and
only cells up to three passages were used throughout the entire experiment.
(Recombinant human BMP-2 and recombinant human bFGF were kindly provided
from Yamanouchi Pharmaceutical Co., Ltd., Tokyo and Kaken Pharmaceutical
Co., Ltd., Tokyo, respectively.)
ALP activity significantly increased at day nine with hMSC combined treatment
of BMP-2 (50 ng/ml) and bFGF (2.5 ng/ml), the cell number was significantly
greater in either bFGF alone or combined treatment of BMP-2 and bFGF by
day seven.
A nude rat (F344/N Jcl-rnu) calvarial defect showed significantly enhanced
bone mineral density four weeks after treatment with hMSC in combination
with either bFGF (100µg) or BMP-2 (10µg) alone or with both
cyto-kine treated, compared to those treated with hMSC alone or phosphate-buffered
saline (PBS) only. Histologically, apparent basophilic mineralization was
observed in hMSC with bFGF alone and partial lamellarity was formed in hMSC
with BMP-2 alone, while the most remarkable bone ridge formation and osteoblast
lining was detected when hMSC treated with a combination of two cytokines
were used. By four weeks, hMSC alone demonstrated basophilic mineralization
and partial lamellarity with a background stromal cell pattern. Bone marrow
space formation and well-structured lamellarity were observed in hMSC with
BMP-2 treatment, while the apparent osteocytes surrounded by an osteoblast
expression pattern were demonstrated by hMSC treated with a combination
of two cytokines. By nine weeks, all treatments demonstrated clear, organized
and mature bone ridge formation.
In a 4x4 cm axial-pattern modified epigastric nude rat flap model, the hMSC
and/or two cytokines in 0.1 ml of phosphate-buffered saline (PBS) were injected
with a 30-gauge needle into the proximal site in the femoral artery with
the distal to the bifurcation of the femoral artery into the epigastric
artery and vein was clamped. All clamps were removed after 10-minute incubation
of the injected agents. The flap was tightly wrapped around a gelatin sponge
and sutured back under the superficial skin. The specimen was investigated
for sequential bone mineral density, histology and osteocalcin expression.
Bone mineral density significantly increased in hMSC treated with bFGF or
a combination of two cytokines by three weeks. By two weeks, faint but clear
bone formation was demonstrated in hMSC treated with a combination of two
cytokines. By four weeks, the bone ridge demonstrated greater density in
hMSC treated with both cyto-kines. The mRNA expression was significantly
enhanced in hMSC treated with both cytokines at two weeks.
BMP-2 and bFGF can synergistically mediate mesenchymal stem cell differentiation
toward bone formation and promote proliferation in vitro and in two in vivo
models. Interaction between mesenchymal stem cells and osteogenic cytokines
was clarified.
Pharmacological Scar Control
Prof Mark Ferguson, School of Biological Sciences, University of Manchester
and RENOVO Limited, Manchester Incubator Building, 48 Grafton Street,
Manchester, M13 9XX, England
Scarring is a major complication following any form of wounding. It can
lead to adverse sequelae of a cosmetic, functional or psychological nature.
Following the observation that wounds on early mammalian embryos heal
perfectly with no scar, we have investigated the cellular and molecular
basis of this scar free healing, comparing events in the embryo with those
in an adult. There are many differences between fetal wounds, which heal
without scarring and adult wounds, which scar, but only a small number
of those differences cause the scar free healing phenotype in the embryo.
Specifically we have focussed on the ratio of growth factors present in
fetal and adult wounds. Fetal wounds have high levels of TGF b3 (present
in developing skin keratinocytes and fibroblasts) and low levels of TGF
b1 and 2 (in the few inflammatory cells pre-sent in the fetal wound, compared
to adult wounds. Experi-mental manipulation of these factors in rodent
and porcine models have shown that exogenous addition of TGF b3 or neutralisation
of TGF b1 and 2 or prevention of the activation of TGF b1 and 2 (by Mannose
6 Phosphate) all lead to adult wounds which heal with markedly reduced
or absent scarring. Dose response experiments have established that the
scar preventing therapy needs to be given at the time of, or shortly after
injury, but has long lasting effects: often years after the initial application.
These therapies have now been developed through appropriate animal models
into the clinical setting. Early investigations on human volunteers in
phase 1 clinical trials are underway and initial results look promising.
We have, therefore, translated these fundamental discoveries into clinical
drug development projects, with the future hope of developing pharmaceutical
agents, which could be used topically, or systemically to markedly reduce
or prevent scarring following all forms of injury.
Effect of Noncontact Normothermic
Wound Therapy on the Healing of Neuropathic (Diabetic) Foot Ulcers
Oscar, M. Alvarez, Ph.D., and Mayank Pate M.D. University Wound Care
Center, Bronx, NY, USA; Roisin S. Rogers, RN, MSN, CWCN and Juanita G.
Booker, RN, BSN, Visiting Nurse Service of New York, New York, USA.
We report findings of a prospective, randomized, controlled study comparing
diabetic foot ulcer healing in 52 patients being treated with either noncontact
normothermic wound therapy* (NNWT applied for one hour three times daily
until healing or 12 weeks) or standard care (control, saline moistened
gauze applied once a day). Surgical debridement and adequate foot off-loading
was provided to both groups. Evaluations (performed weekly) consisted
of acetate tracings, wound assessment and serial photography. Twenty-three
patients were treated with NNWT and 25 patients were in the control group.
Ulcers treated with NNWT had a greater mean percent wound closure than
control treated ulcers at each evaluation point. The relative rate of
healing was significantly greater (P < 0.05) for NNWT treated ulcers
after 4, 6, 8,10 and 12 weeks. After 12 weeks 73% of the wounds treated
with NNWT were healed compared to 37% for the control group (P = 0.019).
The average patient treated with NNWT had a 43% better chance for complete
wound healing than the average patient in the control group. Noncontact,
normothermic wound therapy was safe and easy to use.
*Warm-Up Noncontact, normothermic wound therapy, Augustine Medical
Inc., Eden Prairie, MN
International Clinical Recommendations
on Scar Management
Thomas A Mustoe1 MD (Chairman, USA), Rodney D Cooter2
MD (Australia), Michael H Gold3 MD (USA), FD Richard Hobbs4
FRCGP (UK), AIhert-Adrien Ramelet5 MD (Switzerland), Peter
0 Shakespeare6 MD (UK), Maurizio Stella7 MD (Italy),
Luc Téot8 MD (France), Fiona M Wood9 MD (Australia),
Ukich E Ziegler10 MD (Germany) for the International Advisory
Panel on Scar Management.
1 Northwestern University School of Medicine, Chicago, IL,
USA; 2 Royal Adelaide Hospital & The Queen Elizabeth Hospital,
Adelaide SA, Australia; 3 Gold Skin Care Centre, Nashville,
TN, USA; 4 University of Birmingham, Birmingham UK; 5
Lausanne, Switzerland; 6 Laing Burn Research Lahoratories,
Salisbury District Hospital, Salisbury, UK; 7 Burn Centre,
CTO, Turin, Italy; 8 Burns Unit University Hospital Lapeyronie,
Montpellier, France; 9 Chirurgische Universitatsklinik, Wurzburg,
Germany; 10 Burns Unit, Royal Perth Hospital and Princess Margaret
Children's Hospitals, Perth, WA Australia.
These recommendations were compiled by an international expert panel,
with complete editorial freedom. All panel memhers have collaborated on
this project without remuneration. A small unrestricted educational grant
was provided by Smith & Nephew Medical Ltd and used by the panel for
co-ordination and communication.
Many techniques for management of hypertrophic scars and keloids have
been proven through extensive use but few have been supported by prospective
studies with adequate control groups Several new therapies showed good
results in small-scale trials, but these have not been repeated in larger
trials with long-term follow-up. This paper reports a qualitative overview
of the available clinical literature by an international panel of experts
using standard methods of appraisal. The paper provides evidence-based
recommendations on prevention and treatment of abnormal scarring, and
where studies are insufficient, consensus on best practice. The recommendations
focus on the management of hypertrophic scars and keloids, and are internationally
applicable in a range of clinical situations.
These recommendations support a move to a more evidence-based approach
in scar management. This approach highlights a primary role for silicone
gel sheeting and intralesional corticosteroids in the management of a
wide variety of abnormal scars. The authors concluded that these are the
only treatments for which sufficient evidence exists to make evidence-based
recommendations. A number of other therapies that are in common use have
achieved acceptance by the authors as standard practice. However, it is
highly desirable that many standard practices and new emerging therapies
undergo large-scale studies with long-term follow-up before being recommended
conclusively as alternative therapies for scar management.
Smith & Nephew Group Research
Centre
Presenter: Gareth Lloyd-Jones, Managing Director, Smith & Nephew
Group Research Centre, University of York Science Park, and Honorary Professor
in the Department of Chemistry at The University of York.
'Panel Presentation-Industry Perspective'
The worldwide Medical Devices Industry, currently valued at approximately
$160 billion, continues to grow in importance, primarily due to the significant
changes that are occurring in population demographics along with an ever-increasing
awareness by consumers of changes in medical technology/practice. These
market forces have fuelled increased healthcare costs, which in USA alone
were estimated at $1.3 trillion in 2000.
To ensure real clinical benefits emerge, we have to move away from relying
solely on established technologies. The rapid development of the science
of molecular biology and genetics allows us through better understand
of tissue repair biology to drive technologies that have potential to
build new sectors for the industry.
To be successful in delivering the best solution(s) to the patient, industry
must improve its ability to foster external relationships that will facilitate
adoption and integration of new technologies as a key element of product
development strategy. The value of technology in today's business environment
will be discussed.
'Low Intensity Ultrasound'
One of the most widely used means of effecting biophysical stimulation
of tissue has been ultrasound. Historically 'therapeutic ultrasound' has
been the most extensive form of therapy used clinically principally in
the treatment of pain and musculoskeletal injuries. The application of
therapeutic ultrasound demands constant movement of the device over the
area of injury to avoid tissue damage due to excessive heating. For this
reason the routine application of ultrasound to repair hard tissue such
as bone has until recently been avoided. The observation that low intensity
ultrasound, at levels where energy conversion to heat is judged to be
extremely small, can enhance hard tissue repair has lead to the development
of a low intensity ultrasound device (SAFHS)*.
The considerable body of clinical evidence that now supports the beneficial
effect of this low intensity ultrasound on fracture healing has resulted
in its approval by FDA for use in accelerating healing of fresh fractures
as well as healing of fractures classified as delayed unions.
In vitro and in vivo studies have begun to identify the
effect low intensity ultrasound has at a cellular level and allow some
early insight as to mechanism of action. The preclinical and clinical
evidence in support of low intensity ultrasound therapy for fracture repair
will be reviewed.
*SAFHS - Sonic Accelerated Fracture Healing System; 'Exogen', Smith
& Nephew
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