|
SELECTED
ABSTRACTS
Abstracts
from 14 September (Pisa meeting)
HUMAN EPITHELIAL STEM CELLS IN REGENERATIVE MEDICINE
Graziella Pellegrini1, Paolo Rama2, Fulvio Mavilio3 and Michele
De Luca1,3
1The Veneto Eye Bank Foundation, Venice, Italy; 2H. San Raffaele, Milan,
Italy; 3Dept. of Biomedical Sciences, University of Modena and Reggio
Emilia, Modena, Italy.
Adult stem cells are cells with a high capacity for self-renewal that
can produce terminally differentiated progeny. Stem cells generate an
intermediate population of committed progenitors, often referred to as
transient amplifying (TA) cells, that terminally differentiate after a
limited number of cell divisions. Human keratinocyte stem cells are clonogenic
and are known as holoclones, whilst whilst TA cells are either not clonogenic
or generate aborted colonies known as paraclones.
Corneal regeneration
The human corneal epithelium is suitable to distinguish keratinocyte stem
and TA cells. Corneal stem cells are segregated in the limbus, which is
the transitional zone between the cornea and the bulbar conjunctiva, while
limbalderived TA cells form the corneal epithelium. Human limbal holoclones
can be identified at a molecular level by the expression of the transcription
factor DNp63a. In the uninjured surface of the human eye, DNp63a is present
in the limbus but absent from the corneal epithelium and sustains limbal
cells proliferative potential. DNp63b and DNp63g appear upon wounding
and correlate with limbal cell migration and corneal regeneration and
differentiation.
Cultivated limbal stem cells generate cohesive sheets of corneal epithelium
suitable for clinical application. We report clinical results obtained
in an homogeneous group of over 180 patients presenting with corneal opacification
and visual loss due to chemical burn-dependent limbal stem cell deficiency.
The corneal epithelium and the visual acuity of these patients have been
restored by grafts of autologous cultured limbal keratinocytes.
Gene therapy of Junctional Epidermolysis Bullosa (JEB).
Mutations in genes encoding the basement membrane component laminin 5
(LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and
often fatal skin adhesion disorder. Epidermal stem cells from an adult
patient affected by LAM5-‚3-deficient JEB were transduced with a
retroviral vector expressing b3 cDNA and used to prepare genetically corrected
cultured epidermal grafts. Such grafts were transplanted onto surgically
prepared regions of the patient’s legs. Engraftment was complete
after 8 days. Synthesis and proper assembly of normal levels of functional
LAM5 was observed, together with the development of a firmly adherent
epidermis thatremained stable for the duration of the follow-up (8 months)
in the absence of blisters, infections, inflammation or immune response.
Proviral integration site analysis indicated that the regenerated epidermis
is maintained by a defined repertoire of transduced stem cells. These
data show that ex vivo gene therapy of JEB is feasible and leads to full
functional correction of the disease.
GENETIC APPROACHES TO WOUND HEALING
Jeffrey M. Davidson, Ph.D., Department of Pathology,
Vanderbilt University School of Medicine and Senior Research Career Scientist,
Veterans Affairs
Tennessee Valley Healthcare System, Nashville Campus, Nashville, Tennessee,
USA
There are very few authentic genetic disorders of wound healing, but we
have been empowered with the capacity to control the expression of specific
genes that may be involved in the repair process. This knowledge has permitted
a far more precise interrogation of the biology and pathobiology of repair,
with the promise of identification of critical pathways and modalities
for their correction. Early genetic approaches were designed to over express
gene products previously implicated by protein or RNA studies, and many
classical growth factors and cytokines are now known to be effective as
genetic reagents, often more so because of drug delivery issues. This
strategy has now been extended to deliver not only soluble, intercellular
mediators such as growth factors and cytokines but also intracellular
signals acting in the cytoplasm and nucleus. There are a number of gene
delivery strategies, which vary in efficiency, cost, and potential risk.
Proof of concept will be available soon, since gene transfer for wound
healing is in clinical trials. Gene delivery can also be combined with
cellular therapy and tissue engineering. In a different genetic approach,
transgenic animals can also be used to report specific elements of the
healing response. So-called unbiased approaches to wound mRNA analysis
have led to the identification of novel molecules and pathways that are
modulated during wound healing. It is likely that proteomic approaches
will add a further dimension by identifying and localizing specific gene
products and their post-translational modifications. These data can be
used to develop new genetic or pharmacological strategies. Genetic healing
disorders can be created in the laboratory by intervention, thus teaching
us a great deal about the role of key genes and reminding us of the remarkable
redundancy of the repair mechanism. Tissue- and stage-specific gene deletion
offers a more precise method to evaluate gene losses that would otherwise
be developmentally lethal, and RNA interference could be an alternative
strategy for gene inactivation. With these approaches, we are learning
much more about the fundamental role and the medical application of a
remarkably wide spectrum of genes.
TISSUE
ENGINEERING OF URINARY TISSUE
Gunnar Kratz
Surgical reconstruction of the genitourinary tract has mainly been limited
by the availability of native urothelial tissue. Several alternatives
have been tried over the years such as placental membrane, omentum, bowel
segments and different types of skin flaps. There are significant limitations
with all of these materials such as the frequent formation of fistulas
and strictures due to their incompatibility with the environment in the
urinary tract.
Increased knowledge in cell cultivating techniques and cell biology, in
the field of ‘tissue engineering’, has opened the possibility
of using autologous cultured cells for surgical reconstruction in the
genitourological tract.
Over the years we have developed a method for reconstruction of the urethra
with the use of a three layered cultured autologous tissue. All three
celltypes that are needed are harvested by bladder wash and cultured separately.
To construct an autologous urothelial tissue the three cell types are
combined into an integrated unit with three distinct tissue layers which
is subsequently used in the reconstructive procedure.
We are today using this method in the treatment of boys with hypospadia,
traumatic urethra lesions, bladder exstrophy, as well as in female to
male sex reassignment surgery. The main differences when compared with
conventional methods are a lower frequency of strictures and fistulas,
a more elastic and thereby more functional urethra and a simplified operative
procedure. In this presentation, our experience from the clinical use
of cultured autologous urothelial tissue and our work to ensure the quality
of the cultured transplants will be discussed as well as future perspectives.
ANGIOGENIC
AND LYMPHANGIOGENIC STRATEGIES IN HUMAN DISEASES: WHAT CAN WE LEARN FROM
FISH, FROGS AND MICE ?
Lieve Moons, Flanders Interuniversity Institute
for Biotechnology, Center for Transgene Technology and Gene Therapy, KU
Leuven, Belgium
Revascularization of injured or ischemic tissues with proangiogenic growth
factors may constitute a new approach for tissue repair and for the treatment
of ischemic diseases. However, recent clinical trials with Vascular Endothelial
Growth Factor-A (VEGF-A) had limited success as a result of a narrow therapeutic
window. Therefore, Placental Growth Factor (PlGF) and VEGF-B, two VEGF-A
family members, known to stimulate vessel growth only in pathological
conditions, thereby not causing the side effects of VEGF-A, might constitute
preferred therapeutic candidates.
We evaluated the effect of PlGF and VEGF-B on cardiac function of the
infarcted heart using a murine and a rat model of myocardial infarction.
Our findings show that PlGF and VEGF-B improves cardiac remodelling after
myocardial infarction via stimulation of angiogenesis as well as via direct
effects on cardiomyocytes. In addition, we recently also tested the role
of PlGF in wound healing using a reproducible murine model of excisional
wound healing and found that PlGF was able to accelerate healing of splinted
wounds in genetically diabetic mice. These findings offer new therapeutic
modalities for the treatment of tissue repair in a wide variety of acute
and chronic injuries, particularly in conditions such as diabetes. On
the other hand, developing novel anti-angiogenic strategies to treat diseases
like cancer is still one of the outstanding challenges in the field. Despite
recent successes with VEGFA inhibitors, safer anti-angiogenic drugs, which
can be administered to patients during prolonged periods without the risk
of causing serious side effects, are still needed.
Over the last years, we identified that PlGF contributes to the angiogenic
switch in pathological disorders. Indeed, PlGF correlates with tumor stage,
tumor vascularity, metastasis, clinical symptoms and survival in human
cancer patients. Using K14-HPV16 mice, we obtained results indicating
that PlGF also plays an important role in the development of spontaneous
skin cancers. In addition, a neutralizing anti-mPlGF monoclonal antibody
(mAb), which inhibits PlGF binding to its receptor Flt1, blocks tumor
angiogenesis and growth in syngeneic, subcutaneous B16 melanoma, CT26
colon and orthotopic Panc02 pancreatic tumor models. In addition, preliminary
data also indicate an unexpected role of PlGF in tumor lymphangiogenesis.
These findings may offer novel therapeutic opportunities for the treatment
of cancer and other diseases suffering from excessive angiogenesis. Zebrafish
embryos and Xenopus tadpoles have emerged as powerful small vertebrate
animal models to dissect the molecular mechanisms underlying developmental
processes, by using for example reverse genetic approaches such as morpholino
knockdown or gene overexpression. Moreover, these models are also suitable
for (semi)high-throughput chemico-genetic screens to identify novel target
genes and drugs.
Recently, we characterized the Xenopus laevis tadpole as a useful genetic
tool to study the molecular basis of lymphangiogenesis. Using inhibitor
compounds such as PTK787/ZK, MAZ51 and other inhibitors known to block
receptor tyrosine kinases involved in (lymph)angiogenesis (VEGFR-2, VEGFR-3,
etc), normal development of lymphatics and blood vessels was impaired
respectively in tadpoles and zebrafish embryos. In a further step, by
taking advantage of the unique regenerative capacities of the tail in
Xenopus laevis tadpoles and the fin in adult zebrafish, we have extended
the use of our models for compound screening and lymph/angiogenesis studies
in a condition resembling mammalian wound healing. Amputated tails of
5-day-old tadpoles were found to regrow within a 5- day period, forming
functional blood- and lymph vessels as confirmed by angio- and lymphangiographies.
Initial results showed an inhibition of regenerative lymph/angiogenesis
during tail and fin regeneration by PTK787 and MAZ51. Our findings in
the small animals offer new possibilities for further identifying target
genes or drugs important for tissue repair.
ELEVATED MATRIX-METALLOPROTEINASE ACTIVITY IN CHRONIC
WOUND FLUID
Erin A. Rayment, Gary K. Shooter, Graeme A. George and Zee Upton,
Tissue Repair and Regeneration Program, Institute of Health and Biomedical
Innovation, Queensland University of Technology, Brisbane, QLD, Australia
Aims
There is much debate surrounding the under- and overexpression of MMPs in
chronic wounds. One view is that the overexpression of MMP-3 and MMP-13
correlates with non-healing wounds, whereas active MMPs-1, - 2, -9, and
-14 are all required for normal wound healing.
Others report that elevated levels of MMP-2/-9 are detrimental to wound
healing in prolonged environments, and should be inhibited to allow normal
repair to occur. Further studies indicate that chronic wound fluid includes
high levels of various MMPs, including MMP-1, -2, -3, -8, and -9, leading
to the degradation of key elements in the wound healing process. In this
study we attempt to simplify these findings using a number of MMP detection
strategies and patient samples.
Methods
Chronic wound fluid from six consenting patients was obtained under human
ethical approval. Total protein content was standardized across the samples,
along with human serum and one acute wound fluid sample. Wound fluid was
characterized in the first instance using type I and IV collagen zymography.
In addition, these samples were also incubated with increasing concentrations
of the specific MMP-inhibitor GM6001 (Chemicon, USA) to confirm that the
collagen degrading activity was not due to other proteases. Western blotting
with a number of different MMP antibodies has also been performed to confirm
the presence of specific MMPs.
Results
Type I collagen zymography demonstrates significant differences between
chronic wound fluid and human serum. The elevated levels of MMPs found in
chronic wound fluid also result in slightly different profiles between patients.
Inhibitor studies reveal that increasing concentrations of GM6001 decrease
collagenase activity, leading to complete inhibition of collagen degradation
above a 1 mM concentration. Preliminary western blotting results also show
similar findings.
Conclusions – In future work we wish to:
- Correlate specific
MMP activity with clinical PUSH scores to determine whether a decrease
in activity is directly linked to a healing wound.
- Explore other
MMP inhibitors for clinical applications.
THE
NEUROISCHEMIC DIABETIC FOOT: THE ITALIAN EXPERIENCE
Dr Giacomo Clerici, Diabetic Foot Centre, IRCCS
Policlinico Multimedica, Sesto San Giovanni, Milano.
Mostly, in the 1970s and ’80s, the diabetic foot was synonymous
with ‘mal perforans plantar’ but the lesions mainly related
to the amputations were ischemic ulcers (ulcers and/or gangrene). At the
beginning of the 1980s, LoGerfo published some very innovative and intersting
data: when modern techniques of arterial reconstruction were used, long-term
salvage rates were nearly identical to those in nondiabetics;the term
microangiopathy, therefore, is misleading and should not be used to describe
vascular disease in the diabetic patients. However, the high rate of perioperatory
mortality and cardiovascular disease in the diabetic population, excludes
many people from the possibility of undergoing surgical revascularization,
led us to find a different way to resolve the ischemic diabetic foot problems.
Since the effectiveness of peripheral bypass, including distal bypass
had been established, the role of angioplasty and especially the feasibility
and effectiveness of infrapopliteal angioplasty needed further clarification.
Our studies from the 1990s until today have been done to demonstrate the
feasibility, effectiveness and limits of endovascular revascularisation
in diabetic patients with critical limb ischemia. We demonstrated, with
other groups, that complex arterial lesions, such as lengthy occlusions
of the iliac, femoral, and tibial arteries can often be addressed effectively
by less invasive strategies and today this topic seems to be accepted
in recent guidelines. Eventually, the most important conclusion is that
the use of both, surgical and endovascular revascularisation, improves
the outcome of the diabetic patients and reduces dramatically the amputation
rate. Moreover, the follow up data shows that impossibility of revascularization
because of extent of arterial occlusive disease or because of high surgical
risk is a reliable marker both of a very high risk of above the ankle
amputation and of a very low life expectancy, because of the severity
of the comorbid conditions associated with the severity of CLI.
AMELOGENIN ACTIVITY: IN VITRO DATA
Magnus S. Ågren, Department of Surgery K,
Bispebjerg Hospital, Copenhagen University Hospital, Copenhagen
Amelogenins are hydrophobic extracellular matrix proteins that at physiological
conditions self-assemble into globular aggregates up to micron-sizes. Studies
in periodontal fibroblasts indicate that attachment to these 3-dimensional
structures increases the endogenous secretion of multiple growth factors
and cell proliferation. Topical amelogenins are approved as an adjunct to
periodontal surgical procedures. Recent experimental and clinical studies
indicate that cutaneous wounds also benefit from treatment with amelogenins.
In these studies, the effects of amelogenins on early wound contraction
and promotion of granulation tissue were striking. The mechanisms for these
effects are largely unknown.
Aims:
To study the effects of amelogenins on wound contraction in the dermal equivalent
in vitro model for tissue remodelling and on angiogenesis in the standardized
chick aortic arch assay.
Methods:
In the dermal equivalent model amelogenins (0.01, 0.1, 1 mg/ml) were added
to 72,000 human dermal fibroblasts/ ml in 0.90 mg/ml type I rat tail collagen
prior to collagen polymerisation. Dermal equivalents were subsequently detached
from the culture plates and the area reduction, i.e., contraction, was measured
over seven days. Levels of transforming growth factor-ß1 (TGF-ß1)
and vascular endothelial growth factor (VEGF) in media were measured by
specific ELISA assays. For angiogenic assessments, transverse sections of
the aortic arch of 13-day-old chick embryos were treated with 0.01, 0.1
or 1 mg/ml of amelogenin or with control (17 mM acetic acid). Sprouting
was scored by microscopy by blinded observer.
Results:
In the dermal equivalent model, amelogenins (1 mg/ml) significantly (p <
0.005) increased contraction and fibroblast numbers compared with controls
and also compared with the positive control TGF-ß1 (10 ng/ml). Also,
the levels
of TGF-ß1 and the pro-angiogenic VEGF were increased several-fold
in the presence of amelogenins compared to controls. Amelogenins at 0.1
mg/ml significantly (p < 0.01) increased microvessel outgrowth from the
explants compared with control explants.
Conclusions:
Taken together, these in vitro data indicate that the beneficial effects
of amelogenins on wound healing are, at least in part, mediated by increasing
endogenous growth factor levels (TGF-ß1 and VEGF), that activate fibroblasts
and promote the angiogenic response.
TREATMENT OF INFECTED LEG WOUNDS FOLLOWING SAPHENOUS
VEIN HARVESTING
Adi Zuloff-Shani, PhD1,David Danon, MD1; Arie Orenstein MD2, and Eilat
Shinar, MD1
1) Research and Development Unit, Blood Services Center, Magen David Adom;
2) Department of Plastic Surgery, Sheba Medical Center, Ramat Gan, Israel
Aims
To evaluate the efficacy of local injection of AMS into leg wound infection
following saphenous vein harvesting for CABG surgery as compared to conventional
treatments.
Methods
Patients Between January 2000 and December 2004, 95 patients who underwent
CABG surgery requiring saphenous vein conduit for coronary revascularization
suffering from an infected wound in the incision area, were referred for
AMS therapy (group 1). In the same time frame, conventional treatment of
infected wounds, including Eusol, hydrogels, various dressings, vacuum,
etc., were used in the control group (group 2) for 113 patients also suffering
from infected leg wounds post CABG surgery. All patients were compared,
retrospectively, for response to the treatment they received. Preparation
of AMS A whole blood unit donated by a healthy young volunteer was separated
into red blood cells, white blood cells and plasma. The white blood cells
were treated by hypo-osmotic shock and resuspended in the donor serum to
a concentration of 2 x 106 cells/ml.
Procedure
If necessery, the wound were surgically debrid. Than, the AMS was injected
localy to the wound. Thereafter, sterile gauze pads absorbed with lactated
Ringer’s solution were applied twice daily. This procedure does not
require hospitalization.
Ninty percent of the patients were treated by a single injection of AMS.
No side effects were noted to the treatment. Conventional treatments were
used in the control group according to the manufacturer’s instructions.
Results
In group 1, Ninety-three patients achieved complete wound closure (97.9%)
in a mean of 47 days. In group 2, nintyseven (85.8%) achieved complete wound
closure in a mean of 66.5 days.
Conclusions
We present cell therapy approach for disturbed wound healing process using
AMS prepared from a whole blood unit in a cost effective, sterile closed
system.
COMPARATIVE
EFFECTS OF POLYHYDRATED IONOGENS/HONEY BASED DRESSINGS AND SILVER BASED
DRESSINGS ON THE HEALING OF VENOUS LEG ULCERS.
Tanja Planinsek Rucigaj, Dermatovenerological Clinic,
Clinical Centre, Ljubljana, Slovenia
Introduction:
Wound healing is contingent upon three major factors: bacterial colonization,
production of reactive oxygen species, and the balance between matrix
metalloproteases. Fenolic rich honey with its anti-oxidant properties
is responsible for cleaning and healing wounds; the synthetic blend of
metal ions restores MMP/TIMP balance, inducing epithelialisation. Silver
has known anti-bacterial properties and is widely used to control bacterial
colonization. This study attempts to compare clinical effects of two types
of dressings.
Methods:
Patients with sixty venous leg ulcers (ABPI 0,8 and higher) were included
in stage B1, B2, B3, C1, C2, C3 (Fallanga V. classification of wound bed).
Exclusion criteria were severe disease (cardiac decompensation, rheumatoid
arthritis, uncontrollable hypertension, insulin dependent diabetes mellitus,
carcinoma) and immobility. After approval of the national ethical committee,
patients were randomly positioned into one of two groups by closed numbered
envelopes and subsequently observed for eight weeks or less (if ulcers
were healed). Ulcers in group 1 were treated with honey based dressings
(MelMax®*) and the ulcers in group 2 were treated with silver based
dressings. Compression with long-stretch bandages was used on every patient.
Control visits were after 2, 4 and finally 8 weeks when the study was
closed. At the beginning and end of the study ulcer areas were drawn onto
film dressing and precisely measured by using a digital planimeter**.
Ulcers were classificated into Falanga’s groups.
Results and Discussion:
Ulcers treated with honey-based dressings show similar or faster cleaning
and healing than ulcers treated with silverbased dressings. *Dermagenics
**Placom KP-90N(Japan)
TOPICAL
APPLICATION OF S-NITROSOGLUTATHIONE-CONTAINING HYDROGEL DURING DIFFERENT
PHASES OF WOUNDING IMPROVES RAT CUTANEOUS WOUND REPAIR
Andréa Monte Alto COSTA, Thaís Porto AMADEU, De Souza,
G.F.P2 Amedea Barozzi SEABRA and Marcelo Ganzarolli de-OLIVEIRA, State
University,
Rio de Janeiro, Brazil
Aim:
To investigate the participation of nitric oxide on cutaneous wound repair,
a NO donor (S-nitrosoglutathione, GSNO, 100m) in an hydrogel,
was topically applied on excisional wounds during different phases of
rat cutaneous wound repair.
Methods:
Rats were separated in four groups: a) Control group: in which only hydrogel
was applied during the inflammatory and proliferative phases; b) GSNO
A group: in which hydrogel+GSNO was applied during only the inflammatory
phase; c) GSNO B group: in which hydrogel+GSNO was applied during only
the proliferative phase; and d) GSNO A+B group: in which hydrogel+GSNO
was applied during the inflammatory and proliferative phases. Wound contraction
and re-epithelialization were evaluated. Fourteen days after wounding,
lesions and adjacent skin were collected, formalin-fixed and paraffin-embedded.
Results:
Wound contraction of GSNO A+B group was faster than the other groups 5
and 7 days after wounding. Topical application of GSNO accelerated re-epithelialization
14 days after wounding, mainly in GSNO A+B group. In granulation tissue
of control group, a higher amount of inflammatory cells was observed compared
to GSNOtreated groups. In GSNO A+B-treated group an improvement of granulation
tissue organization and a reduction in the amount of inflammatory cells
were observed. GSNO A+B group showed maturation and advance in granulation
tissue formation. No alterations in mast cell amount as well as in degranulation
of mats cells were observed among groups. Density of myofibroblasts and
vessels was reduced in GSNO A+B group compared to the other groups.
Conclusions:
Nitric oxide is important in all phases of rat cutaneous wound repair,
since topical application of GSNO during two phases (inflammatory and
proliferative) presents better results than application in only one phase
(inflammatory or proliferative). GSNO acts by accelerating wound closure,
wound re-epithelialization and granulation tissue organization. Supported
by FAPERJ, CAPES, CNPq and FAPESP.
BIOAVAILABILITY
OF TGF-BETA AND INDUCTION OF CELL PROLIFERATION IN AUTOLOGOUS PLATELET
GEL PREPARATIONS FOR WOUND CLOSURE
Philipp Huettinger, Thomas Aigner, Nina Brandl and Friedrich Weyer.
Landesklinikum St Pölten Hospital,
Department of Plastic, Aesthetic and Reconstructive Surgery, St Pölten,
Austria; and the Center of Physiology and Pathophysiology, Medical University
of Vienna, Vienna, Austria
Aim:
Cell proliferation, migration and differentiation in wound repair are
governed by cytokines, provided by the platelet containing thrombus. Pivotal
among them is TGF-beta. We here investigated the bioavailability of this
key cytokine in preparations of Platelet Rich Plasma (PRP). We measured
active TGF-beta levels and determined eventual latent forms that, upon
in vitro activation, would enhance the beneficial effect of the PRP.
Methods:
The PRPs were prepared according to the manufacturers recommendations
and assayed for latent and activated forms by ELISA. We used a fibroblast
cell proliferation assay as an indicator for bioactivity of the preparations.The
growth curves in the presence of activated and native PRPs were compared
to control cell cultures. We further demonstrated effectivity of signal
transduction using immunofluorescence microscopy detection of the marker
reaction for TGF-beta, the translocation of the SMAD signalling complexes
to cell nuclei.
Results:
Ratios of latent and activated TGF were found indicative for
an enhanced potency of wound repair. In addition the immunofluorescent
data showed marked differences in the potency to translocate the physiologic
cell signalling from TGF receptor at the plasma membrane to the cell nucleus
in PRP. These in vitro results are correlated with the clinical benefit
of the PRP application on problematic skin lesions with protracted healing
progress.
Discussion:
The results will aid to pinpoint the important factors in PRPs for improvement
of wound healing and may lead to improve the application. Our clinical
experience and result on wound healing using PRP will be presented at
the parallel lecture.
|
