|
YOUNG
INVESTIGATOR AWARDS
ABSTRACTS
Ten
Prize-Winning Oral Presentations
TOPICAL CAPTOPRIL AS A NOVEL AGENT AGAINST HYPERTROPHIC
SCAR FORMATION IN NEW ZEALAND WHITE RABBIT SKIN
GHOLAMREZA S. ARDAKANI and HABIBOLLAH MASOUDI1 – SAEED EBRAHIMI1,
MITRA AMINI1, FATEMEH S. ASLANI2, FARHAD HANDJANP, GHOLAMHOSSEIN R. OMRANI2,
LEILA S. ARDAKANI2, SEYED HAMIDREZA, HOSSEINI ALHASHEMI1 and BEHROOZ KASRAEE1/4
1. Jahrom University of Medical Sciences and Endocrinology; 2. Metabolism
Research Center, Shiraz University of Medical Sciences, Shiraz, Iran;
3. Dept of Dermatology, Shiraz University of Medical Sciences, Shiraz,
Iran; 4. Dept of Dermatology, Geneva University Hospital, Geneva, Switzerland.
Background: Collagen is a protein that constitutes the
majority of extracellular matrix in several tissues such as bone, cartilage
and skin. Over production and/or decreased degradation of collagen fibers
could lead to an abnormal wound healing response resulting in hypertrophic
scar or keloid formation. Angiotensin II has been recently shown to be
present in several cutaneous cells and to stimulate fibroblast proliferation,
collagen synthesis and to suppress matrix metalloproteinase activity.
Herein, we have examined the effect of topical captopril, an inhibitor
of angiotensin II production, against hypertrophic scar formation in New
Zealand white rabbits.
Methods: Three dermal ulcers were made over the ventral
surface of the ears of each rabbit (n = 6). In each animal, separate ulcers
were treated once per day with either topical 5% captopril, the vehicle
alone or triamcinolone acetonide 0.1% cream for seven consecutive days.
Wounds were harvested at post-operative day 28 and the Scar Elevation
Index (SEI) as well as collagen organization were evaluated.
Results: SEI was not significantly decreased by triamcinolone
while this index was reduced from 3.06 in the vehicle treated group to
1.94 in the captopril treated wounds (P<0.05). Triamcinolone, however,
increased collagen organization more than captopril, Le. compared to the
vehicle alone, a 17% increase in collagen organization was achieved by
triamcinolone whereas captopril induced an increase of 8.50%. The increase
in collagen organization was not statistically significant in the case
of each compound.
Conclusion: Results of our study show, for the first
time, the efficacy of topical captopril as a new agent for the prevention
of hypertrophic scar formation. Captopril might thus represent the first
angiotensin converting enzyme inhibitor
with a novel pharmacologic application in dermatology.
Overall Young Inverstigator Prize Winner, S.T.M. Nillesen
(Nijmegen, The Netherlands) with Karin Scharfetter- Kochane
(ETRS President) and Marco Romanelli.
VARIATIONS IN INTRINSIC AGEING PATTERNS AND GENE
TRANSCRIPTION PROFILES OF RESIDENT FIBROBLASTS MAY EXPLAIN SCARRING VERSUS
SCARLESS HEALING
STUART ENOCH, P.E. PRICE, K.G. HARDING, D.V. THOMAS and P. STEPHENS.
Cardiff Institute of Tissue
Engineering and Repair, Cardiff University, UK
Aims: In contrast to skin wounds, oral mucosal wounds
heal rapidly and without scar formation. We have investigated, for the
first time, the onset of senescence in oral mucosal fibroblasts (OMF)
and skin fibroblasts (SF), along with their gene transcription profiles
and phenotypic characteristics, in an attempt to explain these differential
healing responses.
Methods: Patient-matched OMF and SF were cultured until
senescence (determined by telomere length analysis, senescence associated
-β galactosidase activity, cellular morphology and analysis of
population doubling levels).
At early passage (5/6), fibroblasts were synchronised through serum starvation
for 48 hours, re-stimulated with serum and RNA extracted at 0, 1, 6 and
24 hours for transcriptional profile analysis (AffymetrixT Microarrays).
Throughout in vitro ageing, the cellular phenotypes were assessed with
respect to wound repopulation (automated timelapse) and extracellular
matrix (ECM) reorganisation (and involvement of matrix metalloproteinases
[MMPs]).
Results: Compared to SF, OMF progressed rapidly through
cell-cycle (Flow cytometry: 31% of OMF vs. 7% of SF in G2 phase), underwent
more population doublings (>90 vs. 45 in SF) and senesced later. At
early passage, OMF had higher proportion of longer telomeres (p < 0.05;
mean telomere length: SF–4.8 kb; OMF–8.2 kb). Microarray analysis
identified differential regulation of numerous genes between SF and OMF,
some of which were involved in cellular cytoskeleton (Transgelin, Myosin
ID), cell division and cell-cycling (e.g., KLFll/4, GAS6, CyclinG2), and
cell migration and ECM formation (e.g., WISPI, Tenascin XB/ C). K-means
clustering and data annotation demonstrated variations in pathways involving
actin-binding, cytoskeletal protein binding and ECM activity. This was
related to the increased ability of OMF to repopulate in vitro monolayer
wounds and reorganise their surrounding ECM environment (p < 0.05).
Gelatin zymography demonstrated increased production of MM Ps by OMF,
both at early passage (p < 0.01) and at senescence (p < 0.05).
Conclusions: Inherent differences in ageing and gene transcription
profiles identified between OMF and SF may explain the distinct phenotypic
and healing characteristics of these cells. These investigations have
helped decimate some of the mechanics of scarring and scarless wound healing,
and pave the way to develop products/treatments aimed to obviate scarring.
EVALUATION
OF THE USE OF AN IMMUNODEPLETION COLUMN FOR THE PROTEOMIC ANALYSIS OF
CHRONIC WOUND FLUID
M. FERNANDEZ, J. BROADBENT, G. SHOOTER, J. MALDA and Z. UPTON.
Queensland University of Technology, Australia
Aims:
Wound Healing is a dynamic and complex cascade of events. However,
physiological disorders such as chronic leg ulceration have been shown
to be compromised in these vital processes. Since chronic wound fluid
(CWF) itself contains a heterogeneous mixture of proteins and peptides
expressed during healing, we hypothesise that proteins in CWF could be
used as potential markers for healing. In order to explore this, an immuno-depletion
column was evaluated for its ability to selectively remove abundant proteins
shown to mask the presence of proteins in low concentrations in serum.
We specifically evaluated whether this column can be used to efficiently
pre-fractionate CWF, separating high and low abundant proteins for further
proteomic analysis.
Methods: Pooled wound fluid samples were fractionated
using the multiple affinity removal system (MARS) column as per the manufacturer’s
instructions. Two fractions containing high and low abundant proteins
were collected, concentrated and desalted using phenyl reverse phase resin.
Each fraction was evaluated using a wide range of techniques. Firstly,
the selectivity of the column was tested by western blotting using antibodies
against three of the high abundant proteins; albumin; IgG and transferrin.
In addition, 2D gel electrophoresis, 2D proteome fractionation
and mass spectrometry was used highlight the effectiveness of the column.
Results High performance liquid chromatography (HPLC) using the MARS column
resulted in the separation of protein present in CWF into two distinct
fractions. Western blotting and 2D gel electrophoresis demonstrated that
high abundant proteins were selectively removed from CWF resulting in
the enrichment of low abundant proteins. In addition, mass spectrometry
analysis on pre- and post-treated fractions suggested that there was no
protein loss due to interaction of the low abundant proteins with the
proteins removed via MARS.
Conclusions: The results obtained suggest that the MARS
column efficiently removes high abundant proteins enhancing the ability
to detect other proteins in CWF This will improve the separation and identification
of low concentration proteins, potentially allowing the identification
of prognostic and diagnostic markers in CWF.
EFFECTS
OF CULTURED ALLOGENEIC FIBROBLASTS ON THE BASEMENT MEMBRANE ZONE OF IN-VITRO
MODELS OF RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA
L. GAMMON1, T. WONG2, L.M. LEIGH2, J.A. McGRATH2 and H. NAVSARIA1
1. Genetic Skin Disease Group, St John’s Institute of Dermatology,
The Guy’s, King’s College and St Thomas’s School of
Medicine, London; 2. Centre for Cutaneous Research, Institute of Cell
and Molecular Science, Queen Mary Hospital, London.
Aim: Recessive dystrophic epidermolysis bullosa (RDEB)
is an inherited skin disorder caused by mutations in the COL7 Al gene
with a resulting reduction or absence of type VII collagen expression.
Collagen type VII is known to be synthesised in small amounts by fibroblasts
in normal skin although the majority is secreted by keratinocytes at the
dermal-epidermal junction (DEn. Here we used allogeneic, parental and
autologous fibroblasts to try and correct in-vitro models of RDEB.
Method: Combination rafts of RDEB keratinocytes, fibroblasts,
RDEB parental fibroblasts, and normal dermal fibroblasts were created
using de-epidermalised dermis (DED) and collagen gels. Dispase treated
DED rafts were seeded on the reticular surface with fibroblasts and keratinocytes
on the capillary surface. Type 1 collagen gels were infused with fibroblasts
and allowed to contract for two weeks prior to seeding with keratinocytes.
Rafts were raised to the air-liquid interface (ALl) for 12 days before
snap freezing. Cryosections were fixed in acetone/methanol and probed
for laminin 5, collagen type IV and VII. Proliferation assays were preformed
using Alamar Blue over five days and supplemented with ascorbic acid.
Results: Allogeneic fibroblast gels showed diffuse production
of collagen type VII within the gel, but no transport to the DEJ. No staining
was seen with RDEB and parental fibroblasts. Staining for collagen type
IV and laminin 5 showed no difference between all raft combinations. The
proliferation assay showed there was a marked difference between EB fibroblasts
growth compared to normal fibroblasts, with EB fibroblasts proliferating
at a much slower rate. Additionally, low dose ascorbic acid stimulated
proliferation although this was not statistically significant.
Conclusion: Production of collagen type VII from allogeneic
fibroblasts was shown and although not transported to the DEJ may suggest
potential therapies for RDED patients.
VEGF
AND FGF2 INCREASE ANGIOGENESIS AND BLOOD VESSEL MATURATION IN ACELLULAR
SCAFFOLDS FOR TISSUE ENGINEERING
S.T.M. NILLESEN, P.J. GEUTJES, R. WISMANS, J. SCHALKWIJK, W.F. DAAMEN
and T.H.Van KUPPEVELT NCMLS, Radboud University Medical Centre, Nijmegen
Introduction: A major issue in tissue engineering is
the vascularisation of the construct. A strategy to increase angiogenesis
is the incorporation of growth factors. In this study, we test the hypothesis
that two growth factors (VEGF and FGF2) in type I collagen-heparin scaffolds
increase blood vessel formation and maturation in an acellular construct
as opposed to the addition of only one growth factor (either VEGF or FGF2).
Method: Scaffolds consisting of combinations of type
I collagen, heparin, rrFGF2 and rrVEGF were prepared, characterised and
subcutaneously implanted in three-monthsold Wistar rats. At day 3, 7 and
21, explants were retrieved
and histologically evaluated. The explants were stained for type IV collagen
(total blood vessels), smooth muscle actin (SMA; maturated blood vessels)
and hypoxia inducible factor 1-α (HIF1-α, marker for
hypoxia).
Results:
The scaffolds with growth factors revealed more cells than the
scaffolds without growth factors. The staining for COL IV and SMA showed
that the scaffolds with growth factors had a larger number of blood vessels
and more mature blood vessels, especially the scaffold containing both
growth factors. In scaffolds containing growth factors, less hypoxic cells
were found, as shown by HIF1- α staining; least hypoxic cells
were found in the scaffold with both FGF2 and
VEGF.
Conclusion: The addition of two growth factors to a tissue
engineered construct does increase angiogenesis and blood vessel maturation
and decreases the presence of hypoxic cells in the scaffold.
CULTURED
DIABETIC DERMAL FIBROBLASTS EXHIBIT ALTERED PROLIFERATION AND COLLAGEN
SECRETION IN RESPONSE TO WOUNDING AND CIRCULATING FACTORS
S. STEVENSON, L.D. NELSON, P. VOWDEN, D. WHITELAW and M.J. THORNTON.
University of Bradford, UK
Aims: Cutaneous wound healing is impaired in diabetic
patients, which may involve systemic factors. Cellular events critical
to wound healing include dermal fibroblast proliferation and collagen
secretion. We have investigated the response of normal human dermal fibroblasts
and diabetic fibroblasts to mechanical wounding when incubated with non-diabetic
or diabetic human serum.
Methods: Primary cultures of dermal fibroblasts were
established from normal breast skin (n = 5), normal scalp skin (n = 5)
and diabetic leg skin (n = 2). Cell monolayers were left intact or mechanically
wounded, and cultured in the presence or absence of 2% pooled human serum
or individual diabetic sera (n = 9). Proliferation was assessed by 3H-thymidine
uptake and total soluble collagen measured using the Sircol assay.
Results: Diabetic fibroblast proliferation was approximately
twice that of normal fibroblasts. Mechanically wounding significantly
increased proliferation of normal dermal fibroblasts, but had no effect
on diabetic dermal fibroblasts. Basal collagen secretion by diabetic dermal
fibroblasts was three times that of normal dermal fibroblasts. Although
mechanical wounding significantly increased collagen secretion by normal
fibroblasts, it had no effect on diabetic fibroblasts. Normal dermal fibroblast
proliferation was significantly reduced in the presence of diabetic serum
compared to normal human serum. However, no difference in proliferation
was seen when diabetic dermal fibroblasts were cultured with either serum.
In contrast, collagen secretion by diabetic fibroblasts was significantly
reduced in the presence of diabetic serum.
Conclusions: These results suggest that proliferation
and collagen secretion by diabetic and normal dermal fibroblasts differs
in response to mechanical wounding. Normal and diabetic fibroblasts also
demonstrate altered responses to soluble factors in diabetic serum. Since
these factors are inhibitory for dermal fibroblast proliferation and collagen
secretion, these results suggest that impaired wound healing in diabetic
patients is due to both circulating factors and an altered response of
the dermal fibroblast to important wound healing stimuli.
A
PROSPECTIVE STUDY OF AETIOLOGICAL FACTORS AND OUTCOME OF MANAGEMENT OF
LOWER EXTREMITY ULCERS
M. SIBANDA, E. SIBANDA and K. JONSONN,
Dept of Surgery
and Dept of Immunology, University of Zimbabwe College of Health Sciences,
Harare, Zimbabwe.
Background: In Western literature 43–90% of lower
extremity ulcers are due to venous disease. Diabetic patients are particularly
at risk of lower limb ulceration. Delayed wound healing has been noted
in immunosuppressed patients. In sub-Saharan Africa the commonest cause
of immunosuppression is human immunodeficiency virus (HIV) infection.
The frequency of various aetiological factors in patients with lower extremity
ulcers in our setting is not known.
Aim: To prospectively identify aetiological factors and
evaluate outcome of management oflower extremity ulcers in hospital admitted
adult patients, and to assess the impact of immunosuppression on ulcer
healing and limb amputation.
Material and method: A total of 117 adult patients were
recruited for the study after informed consent. Fourteen were excluded
because they had frank gangrene and three patients were lost to follow
up. Thus only 100 patients were analysed. Data was collected by means
of a questionnaire. All patients had full blood count, fasting blood sugar
and serum electrolytes tests done. Sixty-eight patients were tested for
HIV infection by ELISA and 54 of these had T-cell profiles done by flow-cytometry.
Immunosuppression was defined as a CD4+ lymphocyte count of < 500 cell/uL.
Wound size was measured by planimetry. All patients who could stand had
bodymass index (BMI) measured to assess nutritional status. All patients
had their anklebrachial pressure index (ABI) measured by palpation. Statistical
analysis was done by the Epi Info 2002 statistical package. Student’s
Hest was used to test for differences between means and regression analysis
to evaluate independent variables with 95% confidence limits.
Results: The median age was 54 (16–86) years. Infection
(24%) and arterial insufficiency (21%) were the leading causes oflower
limb ulcers. Venous ulcers were rare (1%). Almost half (46%) of the patients
were infected by HIV: More than half of the tested patients (54%) were
immunosuppressed. Nine of these patients had AIDS and 20 more had Category
2 immunosuppression (CD4+ lymphocyte count ranging from 200–499
cells/uL). Diabetes mellitus and HIV infection were independent factors
for immunosuppression. Immunosuppression had no effect on ulcer healing
(p >0.05), but mortality was slightly higher in HIV infected patients
(p = 0.06). Ulcer healing was achieved in 71% of all the patients. Wound
debridement was associated w:ith ulcer healing (p <0.05). Amputation
of the lower limb was done in 9% of the patients. More amputations were
done in patients who either had arterial disease, diabetes mellitus, resided
in a rural area or were smokers (p<0.05). Ten (10%) of the patients
died as a result of their lower extremity ulcers.
Conclusion: Infections and arterial disease were the
leading causes of lower extremity ulcers. Venous ulcers are rare. Diabetes
mellitus and HIV infection independently cause immunosuppression. Almost
half the patients (46%) were infected by HIV. Immunosuppression had no
effect on healing of lower extremity ulcers but may increase mortality.
TIME
DEPENDENT VARIATION OF ANTIMICROBIAL PEPTIDES -BETA DEFENSINS IN BURN
WOUNDS – A KEY TO DESIGN NOVEL ENGINEERED MATRICES TO PREVENT BURN
WOUND INFECTIONS
M. SYED, E. ANTHONY, S. MYERS, P. GREENWELL and H. NAVSARIA
Centre for Cutaneous Research, ICMS, Queen Mary Hospital, University of
London.
The aim of the study was to investigate the expression of Beta Defensins
(BD) 1, 2 & 3 in early (less than two weeks) and late (more than two
weeks) burn wounds and to correlate this to quantitative and qualitative
bacterial inhabitation in these pathologies.
Background: Burn wounds are characterised by colonisation
by variety of resident and pathogenic flora. The colonising and pathogenic
flora is known to become resistant and increase with the duration of the
burn wound. Beta Defensins are inducible antimicrobial peptides produced
by the skin in response to bacterial challenge. It is unclear how the
BD expression varies with the duration of the burn wound.
Methods: Nineteen patients were recruited over a 6-month
period into two groups: burn wounds less than two weeks, and burn wounds
more than two weeks. RNA and Protein expression in burn wounds was analysed
using real time RT – PCR and Immunohistochemistry respectively.
Qualitative and quantitative microbial analysis was performed in both
aerobic and anaerobic conditions.
Results: The expression of all the 3 BD at RNA and protein
stage was significantly decreased in late burn wounds compared to early
burn wounds. BD RNA expression of all the three defensins was elevated
(832 times) in both early and late burns wounds relative to expression
in normal and psoriatic tissue (negative and positive controls). The bacteria
isolated in early burn wounds were: Coagulase negative Staphylococci (49%),
S. aures (19%) Escherichia Coli (13%), Klebsiella (6%), Coryneform species
(13%), and those from late burns were: Coagulase negative Staphylococci
(37%), Methicillin resistant S. aureus (9%), Proteus species (9%), E.
coli (9%) and Pseudomonas species (9%). Thus there was time related shift
towards more pathogenic and resistant flora in burn wounds correlating
to the decreased BD expression.
Conclusion: We demonstrate a change in the expression
profile with time of BD 1, 2 & 3 in burn wounds and its correlation
to the change in the flora. The results of our study will help design
engineered therapies to prevent burn wound infections
A
CLINICAL STUDY INVESTIGATING TYPE VII COLLAGEN EXPRESSION AT THE DERMAL-EPIDERMAL
JUNCTION IN PATIENTS WITH RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA FOLLOWING
INTRADERMAL INJECTIONS OF CULTURED ALLOGENEIC HUMAN FIBROBLASTS
T. WONG, L. GAMMON, L. LIU, L. OZOEMENA, P.J.C DOPPING-HEPENSTAL,
C. JONES, I.M. LEIGH, J.A McGRATH, and H. NAVSARIA.
Genetic Skin Disease Group, St John’s Institute of Dermatology,
The Guy’s, King’s College and St Thomas’ School of Medicine,
London; and the Centre for Cutaneous Research, Institute of Cell and Molecular
Science, Queen Mary Hospital, London.
Aims: Recessive dystrophic epidermolysis bullosa (RDEB)
is an inherited skin blistering disorder due to mutations in the COL7
Al gene leading to reduced/ absent type VII collagen expression and poorly-formed
anchoring fibrils at the dermal-epidermal junction (DEJ). Type VII collagen
is normally synthesised by keratinocytes and fibroblasts and therefore
we assessed whether intradermal injections of allogeneic fibroblasts into
the dermis might lead to increased type VII collagen expression at the
DEJ in subjects with RDEB.
Method: Five RDEB patients (4M, IF; aged 19–39
years) were given intradermal injections of cultured autologous and allogeneic
(P2) fibroblasts (5 x 106 cells in 350–500& #61549;1 of phosphate
buffered saline) and then skin biopsies were taken from each site after
two weeks. Biopsies were assessed by immunohistochemistry for type VII
collagen
expression and for signs of allogeneic cell rejection.
Result: Immunofluorescent staining for type VII collagen
at the DEJ was increased in four of the five individuals. In one patient,
intraepidermal collagen type VII was also observed suggesting potential
paracrine activity from the injected fibroblasts on the overlying keratinocytes.
This individual also experienced mild erythema at the allogeneic cell
injection site although none of the other four patients showed any clinical
evidence of inflammation or rejection. A slight increase in the T helper
cell and macrophage staining were observed in the dermis at injected allogeneic
cell sites compared with autologous cell injection sites.
Conclusion: These initial findings suggest that allogeneic fibroblasts
have the potential to increase type VII collagen at the DEJ in individuals
with RDEB although a mild inflammatory response has been noted histologically.
The subjects in this trial are now being followed up to assess how long
the new type VII collagen will persist at the DEJ as well as the survival
of the allogeneic cells and the host immune response. These preliminary
results may have important clinical implications for a cell-based therapy
for the treatment of RDEB.
APLIGRAF
AS A MODEL OF ACUTE WOUND HEALING: THE EFFECTS OF 17β-ESTRADIOL
AND l,450-NM DIODE LASER ON TYPE III COLLAGEN PRODUCTION
LARISSA L. ZAULYANOV, MD: HUIJUN YUAN, PHD and JEI LI, MD, PHD.
University of Miami, USA.
Background and Objective: Apligraf is a bi-Iayered skin
substitute derived from neonatal human foreskin and bovine type 1 collagen.
The 1,450-nm non-ablative diode laser has previously shown to maximally
increase type III collagen production in this model using 12} of energy.
The purpose of this study is to examine the effect of 17β- estradiol
with and without the addition of 12} of laser energy from the 1,450-nm
diode laser on type III collagen production in Apligraf.
Materials and Methods: First, one sheet of Apligraf was
used to demonstrate staining for the 17β-estradiol receptor using
immunohistochemistry. Next, four sheets of Apligraf were divided into
six groups: control (media only), media with 2nM of 17 β-estradiol,
media with 200nM of 17 β-estradiol, and the aforementioned groups
with the addition of 12} of laser energy. The six groups were incubated
with 10% C02 at 37°C for 3 days and then evaluated for type III collagen
production using RT-PCR. One-way analysis of variance was used for statistical
analysis and a P < 0.05 was considered significant.
Results: Type III collagen production was significantly
increased in three of the groups: the group treated with only 2nM of 17β-estradiol,
the group treated with only 12} of laser energy, and the group that received
the combination of 2nM of 17 β-estradiol and 12} of laser energy.
Although the amount of type III collagen producproduction in the group
receiving 2nM 17 β-estradiol and laser was statistically significant,
the effect was not synergistic. There was no significant increase in type
III collagen production in the group that received only 200nM of 17β-estradiol,
while the group that received this concentration of estrogen with the
addition oflaser did show some increase in type III collagen production,
it also did not reach statistical significance.
Conclusion: Apligraf, similar to normal human skin, expresses
receptors for 17β-estradiol. The 2nM but not the 200nM concentration
of 17βestradiol can significantly increase type III collagen
production in this model. There is no synergistic effect in type III collagen
production with the addition of 12} of energy from the 1450-nm diode laser;
in fact, it appears that the laser and the estrogen induce collagen production
in Apligrafby independent mechanisms.
Three
Prize-Winning Poster Presentations
POLYPYRROLE-BASED CONDUCTING POLYMERS AS SUBSTRATES
IN SKIN TISSUE ENGINEERING
DAVIDSON DAY ATEHCO, PANKAJ VADGAMA and HARSHAD NAVSARIA.
Centre for Cutaneous Research, Institute of Cell and Molecular Science,
St Barts and The Royal London School of Medicine, Queen Mary University
of London
Aims: Polypyrrole (PPy) is a conjugated polymer that
displays unusual electronic properties including conductivity. It may
be electrogenerated with the incorporation of any anionic species including
negatively charged biological molecules such as proteins or polysaccharides.
We have prepared variously loaded-PPy films in order to study the growth
and characteristics of epithelial cells, namely keratinocytes, on these
films and evaluate their potential in tissue engineering.
Methods: PPy films were prepared with various dopants
by electrochemical deposition on gold-coated coverslips (which acted as
working electrodes) using the potentiostatic method. Subsequently, primary
cells and lines were grown on these substrates and studied using biochemical
assays and immunocytochemistry. Furthermore, electrochemical impedance
spectroscopy was studied as an alternative noninvasive means of monitoring
cells on these electrically addressable substrates.
Results: Keratinocyte viability was found to be PPy-loading
dependent. For chloride, polyvinyl sulphate, dermatan sulphate and collagen-loaded
PPy films, polycarbonate and gold, keratinocyte viability as assessed
by the Alamar Blue assay was respectively 47.22%, 60.43%,
87.71 % and 22.65%, 75.44% and 61.04% of tissue culture polystyrene controls
after five days. This was found to require a previously unreported polymer
washing step prior to cell seeding due to the observed toxicity of untreated
films. Keratinocytes stained positive for PCNA (proliferation), KI0 (suprabasal
differentiation) and K16 (hyperproliferation) markers although cell morphology
was poor for organotypical cultures on dermatan-loaded polypyrrole compared
with de-epidermalised dermis. Cell-induced impedance changes were detected
in the three-electrode format over polypyrrole modified electrodes. Results
thus obtained were cell density, cell type and monitoring frequency dependent.
In addition to better cell growth, we found that compared to cells on
gold, there was also an improved resolution for cells on PPy (detection
of lower cell numbers) using this impedimetric technique. Electrical equivalent
circuit analysis in parameters whose contributions may be directly mapped
to the nucleic, cytoplasmic and membrane components of biological cells
showed the technique may be extended to cell type discrimination on PPy
modified electrodes.
Conclusions: This work shows that PPy biocomposites are
attractive candidates for tissue engineering applications since they may
incorporate biomolecules and are electrically addressable with the potential
to both direct and report on cell activities.
STIMULATION
OF CUTANEOUS WOUND HEALING BY REPEATED TOPICAL APPLICATION OF IGF-I: DIFFERENT
MECHANISMS OF ACTION IN RESPECT TO THE IGF-I DELIVERY
S. BECKERT, S. HAACK, H. HIERLEMANN, P. MAYER, F. FARRAHI, A. KONIGSRAINER
and S. COERPER.
Department of Surgery, University Hospital Tübingen, Germany; Institute
for Biomaterials, Denkendorf- Stuttgart, Germany; Wound Healing Laboratory,
University of California, San Francisco, USA
Introduction: Insulinlike growth factor-I (IGF-I) is
accepted to be a potent stimulus of wound healing when applied in combination
with its binding proteins. However, so far no study has been performed
investigating the effect of repeated topical
application of unbound IGF-I. The aim of this study was to show the impact
of daily topical unbound IGF-I therapy on cutaneous ulcer healing in a
steroid suppressed wound model.
Material and methods: Full-thickness wounds were created
on the back of forty male Sprague-Dawley rats. Prior to surgery, animals
received a depot-steroid subcutaneously. Wounds were treated daily either
with topical IGF-I dissolved in 0.2%methylcellulose gel (IGF-I gel) or
with an IGF-I containing hydrogel dressing. After seven days of treatment,
wounds were excised and measured by photoplanimetry. SMA- and PCNA-expression
as well as formation of granulation tissue were assessed in tissue sections.
Results are given as median [min-max]. Differences between groups were
calculated by the Wilcoxon rank test.
Results: Both IGF-I gel (30[27–37] vs. 44[33–65]
mm2; p = 0.0001)and IGF-I dressing (23[12–33] vs. 44[33–65]
mm2; p = 0.0001) enhanced excisional healing as shown by a significant
reduction in wound size with IGF-I released from the dressing being even
more effective than IGF-I gel (p = 0.03). However, only IGF-I released
from the dressing stimulated SMA- (292[196–317] vs. 220[177–
296] pos. cells/mm2; p = 0.03) as well as PCNA-expression (4594[2949–9889]
vs. 3516[2721–4053] pos. cells/ mm2; p = 0.001) and increased granulation
tissue formation (681[513–960] vs. 400[278–714] Ilm; p = 0.018).
Summary: Our data indicate that a daily topical IGF-I
application enhances cutaneous healing. In addition, a controlled
release of IGF-I might stimulate wound repair by different mechanisms
compared with a topical application of IGF-I through methylcellulose gel.
TRANSFECTION
WITH A TETRACYCLIN INDUCIBLE VEGF165 PLASMID RESULTS IN TRANSIENT REGULABLE
HIGH EXPRESSION OF VEGF165 IN VITRO AND IN VIVO
S. DICKENS, P. VERMEULEN MD and H. VRANCKX MD.
Laboratory of Plastic Surgery and Tissue Engineering Research, KU-Leuven,
Belgium
Background: Pathofysiology of non-healing skin wounds
is often related to an altered GF profile. Ex vivo Gene Transfer of Growth
Factors (GF) shows great promise for gene delivery into tissue in a pathological
state. Vascular Endothelial Growth Factor (VEGF) promotes neovascularization
of granulation tissue in skin wounds and therefore seems a good candidate
for gene therapy. However, controlled release of VEGF is obligatory to
prevent hazardous side-effects.
Methods: A hVEGF165 plasmid was ligated into a pcDNA
4T/TO vector using EcoR1 and EcoR5 restriction sites. This results in
h VEGF165 expression under the control of a CMV-promotor and 2 Tetracycline
operator 2 sites (TeT02). Porcine Keratinocytes (KC) were transfected
with pcDNA4/ TO.VEGF (1,5 to 3mg) and pcDNA6T/TR (repression molecule;
9–18mg) by means of lipofectin T in a DNA: lipofectin ratio of 1:4.
For in vitro studies, KC culture
supernatans was collected at timepoints 0, 24, 36 and 48 hours after addition
of 0–5 mg/ml Tetracyclin (TC). For in vivo studies, VEGF transfected
KC are seeded in Full Thickness Wounds (FTW) in a porcine model. Subsequently,
wound fluid is collected at 2, 12, 24, 48 and 72 hours after addition
of 2mg/ml TC. Saline treated wounds are used as negative control. Expression
ofVEGF165 is assessed with ELISA. Biopsies are taken to asses neovascularization
with lectin and anti-CD144 antibodies.
Results: A transient VEGF165 expression is observed in
vitro after TC addition. Peak level is 25,700 pg/ml at 48 hours. When
no TC is added to the medium, VEGF levels remain below 200 pg/ml. In vivo,
VEGF concentration is doubled at t12, 24 and 48 (3464 pg/ ml) in the TC
treated wounds. VEGF levels of FTW without TC and saline treated wounds
remain lower (1673 pg/ml). At t72, VEGF levels of all FTW equal around
6000 pg/ml. An increase in endothelial cells is observed in wounds with
high VEGF expression compared to control wounds (P<0.0001).
Conclusions: A regulable expression of hVEGF165 can be
obtained in vitro and in vivo. The expression is transient in vitro. In
vivo, VEGF levels equal after 72 hours to physiological levels in a porcine
FTW model. High expression of VEGF leads to enhanced vascularization.
|
