Document Sample
					we found focal disruptions along epidermal-dermal junction                             abstracts 56 - 63
only in skin deriving from aged individuals as early as two hours
after loading. To further evaluate changes in ECM we examined
                                                                                       podium Session -
orientation of collagen fibers in dermis using polarized light. We               Wound infection/inflammation
analyzed quantitatively the orientation of the collagen fibers on                Wednesday april 29, 9:15 - 11:30
the basis of the length of the fibers in the plane of the section.
The orientation of the fibers in sections of a loaded aged skin       56
as early as 1/2 hour after loading differed significantly from the    diverSity proFile oF the diaBetic Wound micro-
orientation of fibers of unloaded skin. Focal disruptions in aged     Biome
skin and the change in orientation of collagen fibers identify ini-   Elizabeth Grice, Ph.D.1, NISC Comparative Sequencing Pro-
tial changes that lead to development of pressure ulcers in eld-      gram1, Julia Segre, Ph.D.1, Kenneth Liechty, M.D.2.
erly population. The development of this model will not only          1
                                                                        NIH/NHGRI, Bethesda, MD, USA, 2Childrens' Hospital of
allow for understanding the underlying mechanisms of patho-           Philadelphia, Philadelphia, PA, USA.
genesis, but also allow for pre-clinical, rapid evaluation of po-       The precise relationship between microbes and impaired
tential therapeutic modalities.                                       wound healing remains unclear despite general acknowledge-
                                                                      ment that the resident microflora is a source of complications.
55                                                                    Most microbial surveys of diabetic wounds have relied on cul-
hiF-1α hinderS iSchemic Wound healing                                 ture-based assays even though it’s estimated that >99% of bac-
Jaideep Banerjee, Sabyasachi Biswas, Sashwati Roy, Savita             teria resist isolation in pure culture. We hypothesize that unique
Khanna, Chandan K. Sen.                                               microbial populations and/or shifts in microbial population struc-
Comprehensive Wound Center, The Ohio State University,                ture (the "microbiome") can be detected in diabetic wounds using
Columbus, OH, USA.                                                    less-biased genomic methods. The 16S small-subunit ribosomal
  Bipedicle flaps were developed on the shaved dorsum of mice         (rRNA) genes are universal among prokaryotes and contain
by two full thickness 30 mm long incisions at a distance of 10        species-specific variable regions useful for inferring phylogeny.
mm apart. The incised edges were sealed using electrocautery.         We have previously demonstrated the utility of this method in pro-
A 3 mm circular full thickness punch-biopsy wound was made            filing the bacterial diversity of normal human and mouse skin.
at the center of the flap. Two similar wounds were made in the        Using broad-range PCR, we amplified 16S rRNA genes from the
perfused tissue adjacent to the central wound at the same cran-       excisional wounds of genetically diabetic (db-/db-) mice and non-
iocaudal plane as that of the ischemic wound to serve as con-         diabetic heterozygous controls (db+/db-). 384 near full-length
trols. Incised edges were sutured to the adjacent skin. The flap      16S genes were sequenced from each wound through a time
was imaged by laser Doppler to confirm ischemia. Closure of           course of 28 days. We demonstrate that diabetic mice had a
three (one central ischemic and two laterally non-ischemic            greater bacterial load on their skin prior to wounding. Further-
paired control) wounds were monitored. The ischemic wounds            more, we have identified a unique shift in the bacterial communi-
showed highly significant (p<0.001) impairment in closure. To         ties populating diabetic wounds. These studies are a foundation
test the significance of HIF-1alpha in ischemic wound healing,        for our long-term goal to elucidate the contribution of the diabetic
we stabilized HIF-1a in wounds by adenoviral delivery of HIF-1-       wound microbiome to impaired wound healing.
VP16. To enable the study of HIF-1alpha transactivation in vivo,        This work is funded by the NHGRI Intramural Research Pro-
the wounds were also infected with an adenoviral HRE-lu-              gram and NIDDK Extramural Research Program. E.A.G. is sup-
ciferase reporter construct. HIF-reporter activity was detected       ported by an NIGMS Pharmacology Research Associate
in luciferin injected mice using a IVIS (Xenogen) in vivo imaging     Training fellowship.
system. Images were analysed and luminescent measurements
were performed using the LivingImage software. Delivery of            57
HIF-1-VP16, but not of control VP16, increased HIF transactiva-       inhiBition oF multidrug-reSiStant acinetoBacter
tion. HIF transactivation was significantly higher in ischemic vs.    Baumannii By non-viral eXpreSSion oF hcap-18 in a
control wound (p<0.0001, n=5). Ad-HIF-1-VP16 delivery further         Bioengineered human SKin tiSSue
enhanced (p<0.001) HIF transactivation in ischemic wounds. In         John Centanni1, Christina Thomas-Virnig, Ph.D1, Colette John-
non-ischemic wounds, HIF-1-alpha stabilization caused by Ad-          ston1, Li-Ke He, M.D.2, Sandy Schlosser3, Kelly Van Winkle1,
HIF-1-VP16 did not influence wound closure. In ischemic               Ruibing Chen3, Angela Gibson3, Andrea Szilagyi2, Lingjun Li,
wounds, however, HIF-1 transactivation hindered wound clo-            Ph.D3, Shankar Ravi, Ph.D2, B. Lynn Allen-Hoffmann, Ph.D3.
sure (p<0.05). Topical administration of the HIF-1 stabilizing        1
                                                                        Stratatech Corporation, Madison, WI, USA, 2Loyola University
drug Tilorone also compromised closure of ischemic wounds             Medical Center, Maywood, IL, USA, 3University of Wisconsin,
(p<0.05). These results demonstrate that HIF1a stabilization          Madison, WI, USA.
has an adverse effect on the closure of ischemic wounds. Al-            purpose: Bacterial contamination is the most common reason
though HIF-1a does have angiogenic properties, it is not effec-       for impairment of wound healing. During the healing response,
tive in driving functional angiogenesis in severely                   keratinocytes produce host defense peptides (HDPs) that have
oxygen-deprived tissue.                                               antimicrobial activity against a diverse set of pathogens. The goal
Support: NIH GM077185 and GM 069589.                                  of this study is to genetically engineer a human skin tissue to ex-
                                                                      press the human cathelicidin HDP (ExpressGraft™Enhance) that

W22 2009 SympoSium on advanced Wound care and Wound healing Society meeting
could ultimately be applied to burns and ulcers to counteract bac-       In diabetic foot ulcers, the majority of the wound fluids (>75%)
terial contamination and prevent infection.                            contained both gram +ve and -ve bacteria with predominant types
  methods: A non-viral vector containing the human cathelicidin        being E.coli, S.aureus and Pseudomonas. In these wound fluids
(CAMP) cDNA was used to express cathelicidin (hCAP-18 and              bacterial load correlated with the smaller biologically active form
the mature peptide LL-37) in the NIKS® keratinocyte cell line.         of MMP-9 (actMMP-9 r=0.51, p<0.01) but not the proform. In the
Clonal isolates of stably-transfected cells were isolated. To as-      absence of wound fluid no MMP-9 was present in the bacteria.
sess antimicrobial activity in vitro, topical extracts from Express-   In THP-1 cells addition of bacterial endotoxin from E.coli and S.
Graft™Enhance tissue were incubated with medium containing             aureus increased macrophage proMMP-9 (50 and 20 fold re-
Staphylococcus carnosus. In vivo antimicrobial studies evaluated       spectively) in a dose-dependent fashion. However, only E.coli en-
ExpressGraft™Enhance in a murine burn infection model where full-      dotoxin increased the concentration of actMMP-9. No MMP-9
thickness scald burns were inoculated with A. baumannii. NIKS®         was present in endotoxin from E.coli or S. aureus. Our results
and ExpressGraft™Enhance tissues were grafted on these wounds          showed that bacteria can increase expression of MMP-9 but only
after eschar was removed. Samples were harvested at 72 hrs for         endotoxin from E.coli can increase the activation of MMP-9. How
quantitative bacteriological cultures.                                 E coli increased activation of MMP-9 and the resultant effect on
  results: ExpressGraft™Enhance expresses approximately 140-           tissue turnover remain to be more fully investigated.
fold more hCAP-18/LL-37 than unmodified NIKS® tissue and
possesses key histological features of normal epidermis. Pep-          59
tides extracted from the surface of ExpressGraft™Enhance in-           high percent Body Fat in elective Surgical pa-
hibited growth of S. carnosus by over 78% as compared to               tientS: doeS it increaSe poStoperative inFectionS?
unmodified NIKS® tissue, confirming the antimicrobial prop-            Emily Waisbren, BS1, Heather Rosen, M.D., M.P.H.2, Angela M.
erties of the ExpressGraft™Enhance. Moreover, in an in vivo in-        Bader, M.D., M.P.H.3, Selwyn O. Rogers, Jr., M.D., M.P.H.4, Elof
fected burn wound model, ExpressGraft™Enhance results in a             Eriksson, M.D., Ph.D.1.
two log reduction in a clinical isolate of multidrug-resistant A.      1
                                                                         Plastic Surgery Division, Brigham and Women's Hospital,
baumannii.                                                             Boston, MA, USA, 2Center for Surgery and Public Health,
  conclusions:       Non-viral      genetic       engineering     of   Brigham and Women's Hospital, Boston, MA, USA, 3Anesthesia
ExpressGraft™Enhance alleviates concerns of safety and hetero-         and Center for Preoperative Evaluation, Brigham and Women's
geneity associated with viral transfection. The application of Ex-     Hospital, Boston, MA, USA, 4Division of Trauma, Burns, and Sur-
pressGraft™Enhance to cutaneous wounds will not only provide           gical Critical Care and Center for Surgery and Public Health,
temporary epithelial coverage and growth factors similar to cur-       Brigham and Women's Hospital, Boston, MA, USA.
rently available skin substitute products, but our studies provide       purpose: Studies suggest that obesity is an independent pre-
compelling evidence that ExpressGraft™Enhance is able to combat        dictor of surgical site infection (SSI). BMI is commonly used to
multidrug-resistant nosocomial strains that are of great concern       define obesity. We hypothesized that percent body fat (%BF) pro-
to the medical community.                                              vides a better definition of obesity and is a better predictor of SSI
                                                                       than BMI.
58                                                                       methods: Data from a prospective study of elective surgical pa-
Bacterial load aFFectS Wound Fluid matriX metal-                       tients at one institution were evaluated. Excluded were immuno-
loproteinaSe-9 eXpreSSion and activation: poSSi-                       suppressed, pregnant, transplant, trauma or burn patients for
Ble role in poor Wound healing in diaBeteS                             cohort of 194 patients. %BF was measured using Bioelectrical Im-
Susan V. McLennan, PhD1, Yu Liu, MD1, Danqing Min, PhD2,               pedance Analysis. Preoperative, operative, and 30-day postoper-
Stephen M. Twigg, PhD, MD1, Dennis Yue, PhD, MD1.                      ative outcome variables were obtained using questionnaires and
  University of Sydney, Sydney, Australia, 2Royal Prince Alfred        medical record analyses. The primary outcome variable was SSI.
Hospital, Sydney, Australia.                                             results: Mean age was 48.9 ± 10.2years. Mean %BF and BMI
  Poor wound healing is a common problem for patients with dia-        were 33.8± 10.6 and 29.5± 7.5, respectively. 130 (67%) patients
betes. Our previous studies of wound fluid from diabetic foot ul-      were obese by %BF (males >25%BF, females >31%BF). Only
cers have shown that the initial wound fluid concentrations of         74(38%) were obese by BMI. The overall incidence of SSI was
matrix metalloproteinase MMP-9 and bacterial load are associ-          13.9% (n = 27). Using BMI criteria, 14.2% of non-obese and 13.5%
ated with poor wound healing. The relationship between diabetic        of obese patients developed SSI (p=0.898). Using %BF criteria,
foot ulcer wound fluid bacterial load and MMP-9 is not known           4.7% of non-obese and 18.5% of obese patients developed SSI
and was investigated in this study.                                    (p=0.008). Obesity, defined by %BF, captured 24 of the 27 pa-
  For these studies wound fluids were obtained after debride-          tients with SSI but obesity, defined by BMI, captured only 10. Of
ment from patients (M/F:48/14, age:60.5±10.1yrs, diabetes du-          those with SSI, 88.9% were obese by %BF(p=0.008) and 37% by
ration:14.5±7.5yrs) attending the high risk foot clinic at RPA         BMI(p=0.898). Patients with SSI had significantly higher %BF
hospital for measurement of bacterial load by standard microbi-        than those without SSI (38.5 vs 33.1, p=0.01). However, BMI was
ological techniques and MMP-9 activities by zymography. To ex-         not statistically different between the groups(p=0.1). %BF
amine the relationship between bacterial type and MMP-9                (p=0.01), pedal edema(p=0.05), recent surgery(p=0.05), National
activities, endotoxins were isolated from two common foot ulcer        Nosocomial Infection Surveillance (NNIS) score(p=0.048), and
bacteria (E. coli and S. aureus) and the effect on THP1                wounds with class 2 (clean-contaminated) or higher(p=0.038)
macrophage MMP-9 examined.                                             were univariate predictors of SSI. The C-statistic measured the

                   2009 SympoSium on advanced Wound care and Wound healing Society meeting W23
discrimination of %BF and BMI between patients who will/will not       bit wounds increased the number and duration of macrophages
get SSI. %BF had slightly higher discrimination(c-stat=0.6509) than    in the wound tissue, suggesting that VEGF promotes their disap-
BMI(c-stat=0.6192).                                                    pearance from the wound, likely through stimulation of apoptosis.
 conclusions: BMI may not be the most accurate measurement of          In support of this hypothesis, we found that VEGF decreased sur-
obesity, and %BF is a more sensitive marker of the associated risks.   vival and increased apoptosis in THP-1 macrophages in vitro in a
                                                                       VEGFR1-dependent manner, as shown by increased Annexin V-
60                                                                     FITC staining and caspase activation. We also found that VEGF
Wound macrophage dySFunction in diaBeteS: Sig-                         induces expression of a pro-apoptotic factor, LIGHT, which then
niFicance oF mFg-e8                                                    promotes macrophage apoptosis through activation of the Lym-
Sashwati Roy, PhD, Eric Collard, BS, Sabyasachi Biswas, PhD,           photoxin β Receptor (LTβR); direct activation of LTβR also stim-
Chandan K. Sen, PhD, Savita Khanna, PhD.                               ulates apoptosis, whereas addition of soluble LTβR inhibits
Comprehensive Wound Center, The Ohio State University,                 LIGHT-induced apoptosis. Inhibition of LIGHT-induced signaling
Columbus, OH, USA.                                                     with either anti-LIGHT antibody or soluble LTβR prevented
  Dysregulated inflammatory phase is a key player in the impair-       VEGF-induced macrophage apoptosis, suggesting that VEGF-in-
ment of diabetic wound healing. We have reported that increased        duced LIGHT mediates VEGF-induced apoptosis. To determine
count of apoptotic cells in cutaneous wounds of diabetic mice          the role of LIGHT in inflammation resolution and wound healing
and humans is associated with compromised dead cell clearance          in vivo, we are using LIGHT-/- mice to examine the effect of LIGHT
activity of wound macrophages. Mechanisms of such impairment           deficiency on wound healing. Histological examination of LIGHT-
have not been identified yet. Milk fat globule EGF factor 8 (MFG-      /- wounds shows excessive inflammation in the wound tissue
E8; also known as lactadherin) is secreted by activated                consistent with a critical role of LIGHT in inflammation resolution
macrophages. MFG-E8 acts as a bridging molecule that is capa-          during wound healing in vivo. Our preliminary observations in
ble of binding to phosphatidylserine on apoptotic cells as well as     LIGHT-/- wounds suggest the potential use of the LIGHT defi-
αvβ3 or αvβ5 integrin receptors on macrophages. We hypothe-            cient mouse as an animal model for the study of non-healing,
sized that i) MFG-E8 supports cutaneous wound healing by facil-        chronically inflamed wounds.
itating macrophage dead cell clearance activity; ii) in diabetes
MFG-E8 gets glycated resulting in impairment in dead cell recog-       62
nition by wound macrophages. To address these hypotheses we            local regulation oF cytoKineS With antiBody-
utilized mice lacking MFG-E8 protein. Cutaneous wound healing          FunctionaliZed materialS
was significantly impaired in MFG-E8 null mice compared to the         Newell R. Washburn, Ph.D.1, Liang Tso Sun, B.S.1, Sidi A.
matching wild-type mice suggesting a crucial role of this protein      Bencheriff1, Thomas Gilbert, Ph.D.2, Stephen F. Badylak,
in wound repair process. Wound macrophages isolated from               D.V.M. Ph.D. M.D.2, Michael T. Lotze, M.D.2.
MFG-E8-/- mice were impaired in dead cell clearance as well as         1
                                                                         Carnegie Mellon University, Pittsburgh, PA, USA, 2Univer-
produced increased amounts of pro-inflammatory cytokines               sity of Pittsburgh, Pittsburgh, PA, USA.
(TNFα). We observed that MFG-E8 protein was susceptible to               Inflammation plays a critical role in wound healing process,
glycation. Using ELISA and Biacore assays, we demonstrated             as it prepares the wound site for the subsequent repair mech-
that once glycated the affinity of MFG-E8 for binding with PS was      anism to initiate. However, evidence suggests that inflamma-
markedly diminished suggesting glycation of MFG-E8 in diabetes         tion could lead to scar formation in adult wounds, while fetal
is one of the mechanisms resulting in macrophage dysfunction           wounds, characterized by a relative lack of inflammation, re-
and unresolved inflammation in these wounds. Support: NIH              sult in regeneration and scarless healing. In order to promote
RO1 DK-076566 to SR.                                                   better healing outcome, we hypothesize that native healing
                                                                       trajectory can be altered by modulating inflammation early in
61                                                                     the wound healing process in favor of tissue regeneration. Fur-
light/tnFSF14 promoteS macrophage apoptoSiS                            thermore, recent studies have reported that macrophages, a
in vitro and iS reQuired For normal Wound heal-                        major player in immune system, are able to adopt M1 or M2
ing in vivo                                                            phenotypes in response to stimuli in the microenvironment.
Melissa L. Petreaca, PhD, Manuela Martins-Green, PhD.                  M1 is the predominant phenotype that stimulates inflamma-
University of California, Riverside, Riverside, CA, USA.               tion, while M2 phenotype induces repair by releasing growth
  Wound healing occurs through a series of precisely regulated         factors. Increasing presence of M2 macrophages could poten-
phases, including inflammation, granulation tissue formation, and      tially be beneficial to tissue regeneration by removing factors
remodeling. Any alterations in the regulation of either the initia-    that promote M1 phenotype. In this study, we functionalized
tion or resolution of these phases can impede the healing              hyaluronic acid (HA) with monoclonal antibodies that are ca-
process. For example, many chronic, non-healing wounds exhibit         pable of neutralizing the activities of pro-inflammatory cy-
a prolonged inflammatory phase; inhibition of inflammatory stim-       tokines, including TNFα and IL-1α. In vitro imaging cytometry
uli or stimulation of anti-inflammatory factors may thus facilitate    assay confirmed the same level of IL-1α activity neutralization
healing in chronically inflamed wounds. We have found that Vas-        by the conjugate as that by the unmodified antibodies. In vivo
cular Endothelial Growth Factor (VEGF) promotes inflammation           immunohistochemistry results showed fewer macrophages
resolution through stimulation of Tumor Necrosis Factor Super-         (CD68+) in the wound environment with HA-mAb conjugate
family Member 14 (TNFSF14/LIGHT). Inhibition of VEGF in rab-           treated wounds comparing to pure HA and saline treated

W24 2009 SympoSium on advanced Wound care and Wound healing Society meeting
wounds. About 10 percent of CD68+ cells adopted M1 phe-                           abstracts 64 - 71
notype (CCR7+) in both HA-mAb and pure HA treated
wounds, while saline treated wounds had much higher per-
                                                                                  podium Session -
centage (around 40%) of CCR7+ cells among CD68+ popula-                     gene therapy/Stem cells/etc.
tion. Preliminary in vivo results suggested that the monoclonal             Wednesday april 29, 9:15 - 11:30
antibodies against pro-inflammatory cytokines are able to re-
duce macrophage infiltration to the wound site while HA is ca-     64
pable of promoting M2 phenotype.                                   an inveStigation oF eFFiciency oF gene delivery
                                                                   methodS and time-courSe oF tranSgene eXpreS-
63                                                                 Sion in inJured tendonS and tiSSue reactionS
StaphylococcuS aureuS and epidermidiS Bacte-                       cauSed By diFFerent vectorS
rial BioFilmS delay Wound healing in a novel                       Chuan Hao Chen, MD1, Ya Fang Wu, MD2, Yi Cao, MD2,
model in raBBit                                                    Bella Avanessian, BS1, Xiao Tian Wang, MD1, Paul Y Liu,
Anandev N. Gurjala, MD1, Kai P. Leung, PhD2, Robert D.             MD1, Jin Bo Tang1.
Galiano, MD1, Thomas A. Mustoe, MD1.                               1
                                                                     Roger Williams Medical Center, Providence, RI, USA, 2Af-
  Northwestern Feinberg School of Medicine, Chicago, IL,           filiated Hospital of Nantong University, Nantong, China.
USA, 2Microbiology and Immunology Branch, US Army                    introduction There are a number of ways to deliver gene into
Dental and Trauma Research Detachment, Walter Reed                 tissues. Comparison of different vector system for delivery
Army Institute of Research, Great Lakes Naval Training Sta-        of genes into injured tendon was not reported previously. We
tion, Great Lakes, IL, USA.                                        investigated efficiency of gene delivery to the injured tendons
  purpose: Increasing evidence suggests that bacterial             and tissue reactions caused by different vectors.
biofilms play a critical role in the pathogenesis of chronic         methods Different vectors, plasmid, adeno-associated viral
wounds. The aim of this study was to adapt S. aureus and S.        (AAV), and adenoviral vectors, were used to transfect the in-
epidermidis biofilms to the well-established, quantitative rab-    jured tendon using 72 digital flexor tendons of bilateral toes
bit ear ulcer model, quantitate the effects of biofilm on the      of 18 white leghorn chickens. After transverse tendon cut,
host wound healing response, and investigate the effects of        pCMV-EGFP, pCAG-EGFP, rAAV2-EGFP, and Ad5-EGFP were
a treatment strategy aimed at reducing biofilm burden. We          injected to the tendons. At 3, 7, 14, and 21 days, the tendons
hypothesized that the presence of biofilm elicits a chronic in-    were subjected to examination for GFP expression to deter-
flammatory state leading to a delay in wound healing, and that     mine the efficiency of transgene delivery by different vectors
elimination of biofilm would restore a normal wound healing        under a fluorescence microscope. The tendons were also
response.                                                          stained with hematoxylin and eosin to examine the inflamma-
  methods: Full thickness punch wounds were created in the         tion caused by these vectors. Inflammatory cells were
ears of New Zealand rabbits and inoculated with either S. au-      counted under microscope and were compared statistically.
reus or S. epidermidis biofilm, followed by administration of        results Compared with normal tendons, the GFP expression
both topical bacitracin and systemic enrofloxacin antibiotics      was observed in tendons at 3, 7, 14 and 21 days post-injec-
in order to eliminate planktonic phase bacteria. After 7 days      tion, and was the highest at 7 days for all vectors. At 14 days,
of wound healing, animals were sacrificed and wounds were          we observed a marked decrease in the GFP expression. The
assessed for presence of biofilm and wound healing param-          GFP expression in the tendons injected with rAAV2-EGFP
eters. In a second cohort of animals, wounds with established      and Ad5-EGFP were higher than those with pCMV-EGFP and
biofilm were treated with saline lavage on days 7 - 14 of          pCAG-EGFP vector. No remarkable differences in the GFP
wound healing.                                                     expression were detected between rAAV2-EGFP and Ad5-
  results: Wound beds were visualized using concanavalin A         EGFP vectors. Tissue reactions of the tendons caused by the
immunofluorescence and safranin staining revealing an es-          liposome-plasmid vector (including pCMV-EGFP and pCAG-
tablished biofilm at day 7. Histological analysis yielded in-      EGFP) were the most prominent. Inflammatory reactions of
creased inflammatory infiltrate, and significant detrimental       the tendons with AAV2 vector injected were the least severe.
effects on wound healing, including near total inhibition of ep-     conclusions Efficiency of gene delivery by the AAV2 and
ithelial migration, decreased granulation tissue ingrowth, but     Ad5 vectors is the highest among 4 vectors tested. AAV2 vec-
increased overall granulation tissue area versus control           tor causes the slightest tissue reactions in the tendons. The
wounds. Lavage-treated wounds displayed a reduction in             study suggests that the AAV2 vector is a promising gene de-
biofilm burden, and recovery of wound healing response by          livery vector for tendon gene therapy.
day 14.
  conclusions: Biofilm establishes an inflammatory wound mi-       65
lieu, severely inhibiting epithelialization while exacerbating     induction oF arteriogeneSiS By autologouS Bone
granulaton tissue formation, findings consistent clinical ob-      marroW tranSplantation in critical limB iSchemia:
servations of the chronic wound. Using the rabbit ear dermal       Bonmot-1 and -2 (Bone marroW outcomeS trialS)
ulcer model, we have established an in vivo model which al-        Claas Luedemann1, Jan André Schmidt-Lucke1, Curt Diehm2,
lows for precise quantitation of the biofilm-host response and     Holger Lawall2, Markus Schneider3, Gunnar Riepe3, Bernd
efficacy of anti-biofilm treatments.                               Liesenfeld4, Berthold Amann1.

                  2009 SympoSium on advanced Wound care and Wound healing Society meeting W25
 Franziskus Krankenhaus, Berlin, Germany, 2SRH Klinikum,           (PC-12) up to 5 days (Fig. 1). As low as 4 Gy irradiation re-
Karlsbad-Langensteinbach, Germany, 3StiftungsKlinikum Mittel-      sulted in loss of hematopoietic stem cells and differentiating
rhein, Boppard, Germany, 4Krankenhaus der Barmherzigen             macrophages in the rat bone marrow, on the other hand, the
Brüder, Trier, Germany.                                            mesenchymal stem cells survived in healthy condition. The
  In the US about 150.000 to 300.000 above ankle amputa-           electron microscopic cell morphology of hMSCs in vitro was
tions/year are performed in patients with critical limb is-        maintained with a 60 Gy irradiation. The signaling pathway
chemia (CLI) who can no longer be revascularised. Bone             demonstrated the down-regulation of phosphorylated 90RSK
marrow derived cells can induce arteriogenesis thus improv-        (p90RSK) and subsequent pCREB which lead to the down-
ing perfusion. To prove this concept we started the BONMOT         regulations of VEFG and fibronectin expressions, while the
(BONe Marrow Outcomes Trials) studies. A similar study has         BAD pathways are also down-regulated under Akt (ser 473)
started in the US at 6 centers.                                    regulation.
  In both studies 240 ml of bone marrow is concentrated with        In an immunodeficient nude rat model, artificial dermis appli-
the Harvest SmartPrep® centrifugation system. A bone mar-          cation impregnated with hMSCs and bFGF over the 20 Gy ir-
row nucleated cell concentrate of about 2.30x109 cells is then     radiated skin and soft tissues demonstrated the significantly
reinjected deeply into the limb at 40-60 sites.                    improved fat angiogenesis, architected dermal reconstitution
  In BONMOT-1 (safety and feasibility) 65 CLI patients were        and less inflammatory epidermal recovery. In the mini-pig 10
treated. Primary endpoints were death and rate of major am-        Gy local radiation model, there was distinctive cell population
putation, secondary were wound size, walking distance, pain,       which responds to the external growth factor, a basic fibrob-
ankle-brachial index (ABI), transcutaneous oxygen pressure         last growth factor (bFGF), which stimulates the hMSCs both
(tcpO2). In 60% major amputation could be avoided (expected        in vitro and in vivo, and demonstrated better wound healing
20%). Total wound healing was achieved in 49%. ABI increased       (Fig.2, 3)
within 6 months from mean 0.17 to mean 0.40. TcpO2 from             Mesenchymal stem cells play pivotal roles in radiation wound
mean 10 mmHg to mean 39 mmHg. Absolute walking distance            healing by resisting the radiation itself and via indirect growth
from mean 5 meters (m) to mean 40 m. No procedure related          factor pathway.
side effects occurred.
  BONMOT-2 (launched July 2007) is a double blind, ran-            67
domised, placebo controlled multi center study. Primary end-       epidermal Stem cellS are preServed during
points are major amputation rate and persistence of CLI after      commercial Scale manuFacture oF a Bilayered,
3 months. At the end of April 2009 the three months data of        living SKin SuBStitute (apligraF®) utiliZed For
the first 40 patients of BONMOT-2 can be expected.                 chronic Wound repair
  Until now we have treated the largest collective of CLI pa-      Mark Carlson, PhD1, Katie Faria2, Jon Leman2, Vincent Ron-
tients with autologous bone marrow transplantation (aBMT).         fard2, Jonathan Garlick1.
We have demonstrated that aBMT is a feasible and save              1
                                                                     Tufts University, Boston, MA, USA, 2Organogenesis, Can-
method which can lead to a clinical improvement and limb sal-      ton, MA, USA.
vage in CLI. In the pilot study BONMOT-1 about two thirds of         The purpose of this study was to determine if epidermal stem
the expected major amputations could be avoided. We think          cells (ESC) from neonatal tissue were maintained during the
that the BONMOT-2 study will confirm these results.                commercial scale manufacture of Apligraf®, a human bilay-
                                                                   ered skin substitute (BLSS). To accomplish this, we geneti-
66                                                                 cally marked replicating cells in 2D-culture with a
role oF meSenchymal Stem cellS in radiation in-                    Beta-galactosidase (β-gal)-expressing retrovirus and moni-
JurieS                                                             tored their fate after incorporation into BLSS and subsequent
Sadanori Akita, MD, PhD, kozo Akino, MD, PhD, Naoshi Ki-           in vivo transplantation to a nude mouse. Previous studies have
noshita, MD, Akiyoshi Hirano, MD, PhD, Shunichi Ya-                shown that ESCs are spatially clustered in hierarchical units
mashita, MD, PhD.                                                  known as epidermal proliferation units (EPU). In our study, his-
Nagasaki University, Nagasaki, Japan.                              tological analysis of tissues showed β-gal-positive cells scat-
 Even though detailed mechanisms of the intractable and pro-       tered throughout the epithelium before grafting. Eight weeks
longed wounds after irradiation, there are clinical evidences      post-grafting, smaller numbers of β-gal -positive cells were
that the local mesenchymal stem cells are expressed in the         present in the tissue and distinct β-gal -positive clusters simi-
base of the intractable radiation injured wounds. Therefore,       lar to EPUs were visible 16-32 weeks after grafting.
we sought to investigate the role of mesenchymal stem cells          32 weeks after grafting, cells were isolated from the grafts
in radiation wound healing.                                        and expanded in vitro (2D). Colony forming efficiencies (CFE)
 Human mesenchymal stem cells (hMSCs) are first tested cell        for recovered cells were then compared to the original pre-
growth and signal transduction pathway after irradiation, then     graft population of β-gal -positive cells. The CFE of the cells
both small and large animal model were used for treatment          regrown from the grafts was similar to pregraft CFEs (45% and
and assessment of the radiation wounds.                            40%, respectively) while the proportion of β-gal -positive ESC-
 The hMSCs are strongly proliferative even after 20Gy irradi-      like colonies recovered from grafted Apligraf® was also simi-
ation in vitro in contrast to the cell death of other human neu-   lar to the pregraft tissue (7% and 10%, respectively). Cells
roblastoma cells (NG108-15) and rat pheochromocytoma cells         expanded from the grafts were incorporated into new BLSS

W26 2009 SympoSium on advanced Wound care and Wound healing Society meeting
and showed full differentiated epithelium. These findings pro-     69
vide clear evidence that progenitor ESCs were sustained dur-       compariSon oF eFFicacy oF preFaBrication oF
ing the tissue fabrication and transplantation.                    the iSchemic FlapS With aav2-mediated vegF and
 This study demonstrate that the process of isolation and ex-      BFgF gene therapy
pansion of cells in Apligraf® construction is an enabling tech-    Xiao Tian Wang, MD1, Bella Avanessian, BS1, Chuan Hao
nology that retains viable stem cells. These ESCs organize into    Chen, MD2, Ya Fang Wu, MD2, Jin Bo Tang, MD1, Paul Y
EPUs in vivo, have similar CFEs in vitro to the pregraft popu-     Liu, MD1.
lation, and are able to form a new BLSS that exhibits normal       1
                                                                     Roger Williams Medical Center, Providence, RI, USA, 2Affil-
architecture and differentiation. The presence of ESCs in          iated Hospital of Nantong University, Nantong, China.
Apligraf® may contribute to a robust therapeutic product that        introduction: In reconstructive surgery, necrosis of surgically
functions to heal chronic wounds.                                  transferred flaps is a major problem often resulting in a hard-
                                                                   to-treat wound. A number of gene therapy approaches, with
68                                                                 varied success rates, have been attempted experimentally to
lentiviral mediated il-10 overeXpreSSion pro-                      combat this problem. In this study, we evaluated the efficacy
moteS ScarleSS Wound healing Without aFFect-                       of prefabricating ischemic flaps with AAV2-medicated VEGF
ing Wound BiomechanicS                                             or bFGF gene transfers in promoting the survival of the is-
Benjamin J. Herdrich, MD1, Dustin M. Bermudez, MD1,                chemic flap.
David P. Beason, MS2, Louis J. Soslowsky, PhD2, Kenneth              methods: Twenty-seven Sprague-Dawley rats were divided
W. Liechty, MD1.                                                   (7 to 10 rats per group) into two experimental groups and one
  University of Pennsylvania School of Medicine and the            non-treated control group. 2.9 x 1010 AAV2-VEGF or 7.5 x 1010
Center for Fetal Research at the Children's Hospital of            AAV2-bFGF viral particles were injected into the dorsum of
Philadelphia, Philadelphia, PA, USA, 2University of Pennsyl-       each of the rats of the two experimental groups. Two weeks
vania School of Medicine and the McKay Orthopaedic Re-             post-injection, a 3 x 7 cm flap was raised from the injection
search Laboratory, Philadelphia, PA, USA.                          area. In the control group, this flap was raised with prior pre-
  introduction: Fetal regenerative healing proceeds with mini-     fabrication. One week after post-surgery, flap viability was
mal inflammation, regeneration of dermal elements, and lack        measured. In histologic sections, vascularization and immuno-
of fibroplasia. We have previously shown that overexpression       histochemical staining of VEGF or bFGF were evaluated.
of the anti-inflammatory cytokine IL-10 leads to a diminished        results: Flap viability was significantly improved by the
inflammatory response and regenerative healing in the adult.       AAV2-VEGF prefabrication (p<0.01). The greatest survival
It is not known whether this has an effect on the quality of the   area among all samples was in a flap prefabricated with AAV2-
wound repair. We hypothesized that lentiviral mediated IL-10       bFGF; however, the mean viability for the AAV2-bFGF prefab-
overexpression would not impair the biomechanical proper-          rication group was equivalent to that of flaps prefabricated
ties of the healed wound.                                          with AAV2-VEGF. Despite this similarity, the flap viability of the
  methods: 8-10 week old C57Bl/6 mice were shaved and pre-         AAV2-bFGF group was not statistically different from that of
treated on their dorsal skin with either lentivirus expressing     the non-treatment group, because of a wide standard devia-
IL-10 and GFP (lenti-IL-10) or GFP alone (lenti-GFP). Three        tion. Histologically, vascularity was increased in two prefabri-
days later, 4mm excisional wounds were created at the injec-       cation groups; immunohistochemical staining showed greatly
tion site, and the wounds were allowed to heal for 4 weeks be-     enhanced VEGF or bFGF expression.
fore they were harvested. A uniform specimen was stamped             conclusions: Ischemic flap prefabrication with AAV2-VEGF
out of each sample which included the wound site. Material         more consistently improves flap survival as compared to pre-
properties of each sample were measured including cross-           fabrication with AAV2-bFGF. Prefabrication with AAV2-bFGF
sectional area, maximum stress to failure, and modulus. The        both effectively increases flap vascularity and also increases
data were analyzed using an unpaired, 2-tailed t-test with         mean survival areas, but it does not lead to constant improve-
p<0.05 taken to denote significance.                               ment in flap survival. Further optimization of used methods or
  results: Lenti-IL-10 and lenti-GFP treated dermal wounds         dosages of AAV2-bFGF may be necessary in future studies.
showed similar cross-sectional areas at 4 weeks (3.44±0.82
mm2, n=9 vs. 3.02±0.89 mm2, p=0.35). In addition, lenti-IL-10      70
and lenti-GFP treated dermal wounds had similar maximum            hypoXia enhanceS eX-vivo eXpanSion oF multipo-
stress to failure (0.50±0.25 MPa, n=9 vs. 0.60±0.22 MPa, n=7,      tential Stromal cellS
p=0.42) and modulus (2.89±1.85 MPa, n=9 vs. 2.71±1.64 MPa,         Kenichi Tamama, MD, PhD, Savita Khanna, PhD, Sashwati Roy,
n=7, p=0.85) at 4 weeks.                                           PhD, Chandan Sen, PhD.
  conclusions: Lentiviral mediated IL-10 treatment promotes re-    The Ohio State University Medical Center, Columbus, OH, USA.
generative dermal healing but does not impair the biomechanical     introduction: Cell therapy with bone marrow multipotential
properties of the healed wounds. This further supports the study   mesenchymal stromal cells (MSCs) is a very promising ap-
of strategies to modulate the inflammatory response to promote     proach to promote wound healing and tissue regeneration.
regenerative healing and minimize scar formation in disease        However, the limited number of collected MSCs from the body
states where fibrosis is a pathogenic feature.                     necessitates the in vitro expansion of these cells after cell har-
                                                                   vest, and it must be conducted in a way to preserve the undif-

                  2009 SympoSium on advanced Wound care and Wound healing Society meeting W27
ferentiated phenotype with multipotent differentiation poten-      71
tials even after long passages for optimal cell therapy with       epidermal activation in SKin But not mucoSa
MSCs. Hypoxic condition is known to prolong replicative life       FolloWing inJury: a potential mechaniSm For
cycle and to help maintain undifferentiated phenotype in other     the regulation oF Scarring
types of cells. Hypoxia inducible factor (HIF) pathway is a cru-   Corrie Gallant-Behm, PhD, Nakshatra Roy, PhD, Thomas
cial signaling pathway activated in response to limited O2 ex-     Mustoe, MD.
posure, and we hypothesize that HIF signaling pathway would        Northwestern University, Chicago, IL, USA.
play a pivotal role in the hypoxia-mediated enhancement of ex        purpose: Cutaneous scarring is a major clinical problem. In
vivo expansion of MSC.                                             contrast, there is minimal scarring in mucosa, a finding which
  material and methods: MSCs were purchased from Lonza.            may provide important insights into the mechanism(s) regulat-
Replicative life span was assessed by serial determination of      ing fibrosis. As the epidermis differs morphologically in skin
cell count up to 40 days. HIF signaling in MSC was studied         and mucosa, we explored the hypothesis that the epidermis
using a HRE-reporter assay and immunoblotting of stabilized        plays a critical role in dictating scar formation.
HIF. We determined colony formation by manual count, os-             methods: A 2x2cm grid of partial-thickness incisions was cre-
teogenic differentiation by alkaline phosphatase activity, and     ated on the ventral surface of the rabbit ear, and equivalent in-
paracrine activity by cytokine array (Raybiotech) and ELISA.       cisions were made longitudinally in the vagina of the same
  results and conclusion: HRE-reporter assay showed the            animal. Wounds were harvested at 12 hours, and 1, 3, and 5
strong activation of HIF signaling in MSCs cultured in 1%O2        days after injury. Histological analysis was performed, and
condition, not in 20%O2 condition. Immunoblot analysis             total RNA was selectively isolated from the epidermis of both
demonstrated stabilization of HIF-2α in response to limited O2     tissues. Quantitative RT-PCR was performed for selected cy-
exposure, whereas that of HIF-1α was minimal. MSCs cul-            tokines and growth factors.
tured in 1%O2 condition had more colony formation, longer            results: All mucosal wounds and 50% of the cutaneous
replicative life span, and stronger secretions of various growth   wounds were re-epithelialized by 24 hours after surgery. Sig-
factors and cytokines than in traditional 20%O2 culture con-       nificant epidermal hypertrophy was observed in the cuta-
dition. Osteogenic differentiation was reversibly inhibited by     neous wounds at 3-5 days post-injury, but not in the mucosal
1%O2 exposure. These actions were reproduced by HIF stabi-         wounds. Molecular analysis demonstrated significant differ-
lizer dimethyloxaloylglycine (DMOG) or reversed by shRNA           ences in gene expression for all cytokines and growth factors
against HIF-1β. These data suggest the significant role of HIF     measured. IL-1β and TNF-α were more highly upregulated in
signaling in the hypoxia-mediated enhancement of ex vivo ex-       skin than mucosa (IL-1β: 46-fold vs. 19-fold, TNF-α: 7-fold vs.
pansion of MSCs.                                                   2.6-fold), and this upregulation persisted for a longer period
                                                                   of time. TGF-β1 had a greater upregulation in skin, over the
                                                                   same time course as mucosa (10-fold vs. 2.5-fold). TGF-α was
                                                                   upregulated in skin, and downregulated in mucosa.
                                                                     conclusions: The cutaneous epidermis is highly activated fol-
                                                                   lowing even a minor injury. In contrast, the vaginal epidermis
                                                                   demonstrates a lesser degree of activation, and more quickly
                                                                   returns to a baseline state. Thus, the epidermis likely plays a
                                                                   crucial role in controlling the formation of a dermal scar, as
                                                                   many of the cytokines and growth factors produced by ker-
                                                                   atinocytes have been shown to directly stimulate proliferation
                                                                   and collagen production by dermal fibroblasts.

W28 2009 SympoSium on advanced Wound care and Wound healing Society meeting
  Wound Healing
     Societ y
19 t H a n n ua l M e e t i n g
 P o S t e r a b S t r ac t S

2009 SympoSium on advanced Wound care and Wound healing Society meeting W29
               abstracts 72 - 106                                       skin substitute specifically designed to promote robust vasculariza-
                                                                        tion and healing of chronic wounds by expressing elevated levels of
   poster general Session - Blue ribbon posters                         angiogenic factors.
           monday april 27, 7:30 - 9:00                                   methods: The well-characterized NIKS® keratinocyte cell line was
                                                                        stably transfected with non-viral plasmid vectors encoding either a
72                                                                      single angiogenic factor (VEGF) or a transcriptional regulator that
conneXin43 carBoXyl-terminal peptideS reduce in-                        induces multiple angiogenic factors in response to hypoxia (HIF-1α).
Flammation and Scar progenitor tiSSue FolloWing                         Skin substitute tissue prepared from the modified cells (Express-
SKin Wounding.                                                          Graft™Vascular) was evaluated for transgene expression and se-
Gautam S. Ghatnekar, PhD1, Michael P. O'quinn, PhD2, Linda              cretion      of    angiogenic       factors.     Vascularization      of
Jane Jourdan2, Abhijit A. Gurjarpadhye, MS2, Robert Draughn,            ExpressGraft™Vascular tissue was quantified by CD31 staining
PhD2, Robert G. Gourdie, PhD2.                                          nine days after engraftment on nude mice.
 FirstString Research, Inc., North Charleston, SC, USA, 2Medical          results: Clonally-pure isolates of stably-transfected cells were iden-
University of South Carolina, Charleston, SC, USA.                      tified that each had the expression vector integrated at a single site.
  Gap-junctional connexin43(Cx43) has roles in multiple aspects of      Genetically-modified skin substitutes expressed VEGF at levels 10-
cutaneuous healing including injury signal spread, leukocyte extrava-   20 fold higher than unmodified tissue and secreted elevated levels
sation, wound closure, granulation and fibrosis. We previously de-      of several pro-angiogenic CXCL chemokines (CXCL1, CXCL5, and
scribed a peptide incorporating carboxy-terminal (CT) amino acids       CXCL6). Conditioned medium from ExpressGraft™Vascular tissue
of Cx43 that has quantifiable effects on connexon clustering, phos-     stimulated the proliferation of human microvascular endothelial cells
phorylation and organization. Prompted by observations in a fibrob-     compared to medium from unmodified skin substitutes. Following
last "scratch-wound" assay, we investigated the uptake and effects      skin substitute engraftment onto nude mice, the dermal region un-
of Cx43 CT peptides on healing following skin wounding in mouse         derneath ExpressGraft™Vascular was vascularized to a greater de-
and pig. Relative to controls, Cx43 CT peptide-treated excisional       gree than wounds grafted with unmodified tissue. Importantly, the
wounds on mice demonstrated decreased inflammation (i.e., red-          resulting vasculature showed no evidence of abnormal vessel per-
ness/swelling (p<0.001) and myeloperoxidase-positive neutrophil         meability.
density (p<0.03)), improved scar appearance (p<0.001), and en-            conclusions: ExpressGraft™Vascular skin substitute tissue pre-
hanced closure rate (p<0.001). Peptide-treated wounds on mouse          pared from a consistent, homogeneous keratinocyte source stably
and pig at day-10 and day-30 post-injury showed decreased granu-        modified with a non-viral vector offers significant advantages in prod-
lation areas (p<0.001) and increased epidermal rete peg densities       uct safety and uniformity compared to products prepared from vari-
(p<0.01). Tensile testing of healed mouse scars at day-90 post-injury   able keratinocyte populations heterogeneously modified with viral
indicated increased stiffness (p<0.005) and modulus-of-elasticity       vectors. The ability of skin substitutes expressing elevated levels of
(p=0.06) and stress-(p<0.03) and strain(p<0.001)-at-rupture relative    angiogenic factors to promote increased vascularization in vivo sup-
to controls. At day-30 post-injury, granulation tissue of peptide-      ports the hypothesis that these substitutes may accelerate the vas-
treated pig wounds demonstrated patterns of sub-epidermal vascu-        cularization and healing of chronic wounds.
larization and densities of alpha-sma+ myofibroblasts (p<0.001) that
were quantifiably similar to unwounded skin, but not to controls. In    74
conclusion, Cx43 CT-peptides reduce inflammation, scarring and          matriX delivered KeratinocyteS: potential rapid
loss of tissue architecture/function following cutaneous wounding       treatment For Severe BurnS
and may have applications for regenerative healing in other injuries    Eugene Skrabut, MS, Grace Kielpinski, BS, Jacqueline Wolfrum,
including those to the cornea and heart. This research work was sup-    PhD, Sandra Dethlefsen, PhD, Bruce Wentworth, PhD.
ported by NIH grants (NIH, HL56728 and 1R41AR053798 to R.G.G.           Genzyme Biosurgery, Cambridge, MA, USA.
and 1R43DK080567 to G.S.G.). R.G.G. is supported by MUSC                 Severely burned patients are successfully treated with sheets of
Board of Trustees’ Eminent Scholar award.                               cultured epidermal autograft (CEA). This process is life-saving even
                                                                        with a three to four week preparation time from the receipt of a
73                                                                      biopsy to the availability of the product for the patient. CEAs provide
a human SKin SuBStitute engineered uSing a non-                         only the epidermal layer of skin and are susceptible to shear forces
viral vector SecreteS elevated levelS oF angio-                         which can lead to a lower engraftment take. While CEAs provide
genic FactorS and enhanceS vaSculariZation in                           permanent coverage, the mechanical properties of the closed
vivo                                                                    wound are not optimal. An optimal product design might enable a
Allen Comer, Ph.D.1, Joely Straseski, Ph.D.2, Sara Pirnstill, B.S.1,    more rapid delivery of a subconfluent epidermal graft by seeding
Kristi Schaeve, B.S.1, Maggie Bach, B.S.1, Jennifer Murphy, M.D.2,      cells onto a rugged biocompatible matrix that could also serve as a
Lynn Allen-Hoffmann, Ph.D.1.                                            dermal substrate and provide a permanent, mechanically durable
 Stratatech Corporation, Madison, WI, USA, 2University of Wis-          closure. The product might facilitate attachment to the wound bed,
consin School of Medicine and Public Health, Madison, WI, USA.          enhance vascularization, minimize scarring at the margins, and re-
  purpose: Current skin substitutes rely on expression of endoge-       duce wound contracture. We describe here the preparation of sub-
nous factors and are not designed to address the poorly-vascular-       confluent epidermal grafts on a collagen matrix delivery system.
ized, hypoxic wound environment that impairs the healing of chronic      Cryopreserved human keratinocytes were expanded in culture,
wounds. The goal of this study is to develop a second generation        harvested, resuspended in tissue culture medium and seeded at a

W30 2009 SympoSium on advanced Wound care and Wound healing Society meeting
density of (2x105/cm2) onto a collagen matrix without xeno-                    ON, Canada, 3Marine Polymers Technologies, Inc., Boston, MA,
geneic feeder cells. After three days, 8mm punch samples were                  USA, 4Sunny Brook Research Institute, Univeristy of Toronto,
taken for scanning electron microscopy (SEM), histology, and                   Toronto, ON, Canada.
metabolic activity testing (alamar blue reduction and thiazolyl                  Recent findings show that treatment of cutaneous wounds with
blue (MTT) staining).                                                          poly-N-acetyl-glucosamine nanofibers (pGlcNAc), a novel polysac-
  Good cell viability and metabolic activity were demonstrated utiliz-         charide material derived from a marine diatom, results in an in-
ing the alamar blue assay and MTT staining, respectively. SEM                  creased kinetics of wound healing that can be attributed, in part, by
showed the cells were attached and extensively spread along the                a marked increase in angiogenesis. Our published data suggests
membrane surface and within the pores of the collagen membrane.                that treatment of primary endothelial cells (EC) with this nanofiber
Five micron paraffin cross sections of the membrane stained with               results in a marked increase in cell migration, which is due to an in-
hematoxylin and eosin showed cells attached to the membrane sur-               tegrin-dependent up-regulation of the Ets1 transcription factor. We
face, as identified by the presence of nuclei. This study suggests the         show that pGlcNAc stimulation of Ets1 results from the activation
feasibility of delivering keratinocytes to the patient within three to         of Akt1 by these nanofibers. Nanofiber treatment of EC also results
four days of seeding onto a collagen matrix, without the need for              in marked increases in the metabolic activity of EC and in the expres-
xenogeneic feeder cells.                                                       sion of genes involved in cellular recruitment, such as IL-1 (a known
                                                                               Ets1 target), VEGF and several defensins (α3, α1, α4, and α5), small anti-
75                                                                             microbial peptides recently shown to act as chemo attractants. Phar-
development oF an epidermal SKin eQuivalent in                                 macological inhibition of the Akt1αEts1 pathway results in decreased
vitro Wound model                                                              expression of these chemotactic factors. Our results suggest that
Brett Kiedaisch, Tasha Garcia, Eric Rolland, Dennis Carson, Dun-               nanofiber-activated endothelial cells secrete factors that may influ-
can Aust.                                                                      ence the recruitment of other cell types such as lymphocytes, ker-
HealthPoint Ltd, Fort Worth, TX, USA.                                          atinocytes or fibroblasts required for wound healing. To test this
 The development of full-skin, epidermal, and dermal substitutes               hypothesis, nanofiber treated EC were used in modified transwell
have been very valuable in the advancement of wound care. in vitro             chemotactic assays. We find that nanofiber treatment of EC results
skin equivalent models have been used to investigate many aspects              in a marked increase in the recruitment of fibroblasts toward pGlc-
of wound repair including fibroblast proliferation, tissue remodeling,         NAc-activated EC and that blockade of the Akt1αEts1 pathway in-
and growth factor and cytokine secretion1,2. The 3D nature of these            hibits this recruitment. Taken together our findings suggest a central
skin substitutes provides a more dynamic environment that closely              role of the Akt1αEts1 pathway in the regulation of cutaneous wound
resembles actual wound beds. Compared to 2D or monolayer mod-                  healing by pGlcNAc nanofibers and support the use of these fibers
els cells within skin substitutes more closely behave and interact             as a novel and effective method for enhancing wound healing.
with the extracellular matrix as they would in vivo2.
 We have developed an in vitro wound model using an epidermal                  77
skin equivalent derived from the outer root sheath (ORS) cell from             digeStion productS releaSed From mmp treated
hair follicles. After 14 days in culture a well differentiated, mitotically,   Small inteStine SuBmucoSa Wound matriX and
and metabolically active epidermis is grown containing well defined            their eFFect on Keratinocyte migration
basal, spinous, granular, and cornified layers. Wounding of the epi-           Ryan Ermis, BS1, Tasha Garcia1, Dale Telgenhoff, PhD2, Lei Shi,
dermal layer was accomplished using either a scalpel for smaller               PhD1.
wounds or a 2 mm biopsy punch for slightly larger and more repro-              1
                                                                                Healthpoint Ltd., Fort Worth, TX, USA, 2Tarleton State University,
ducible wounds. Once wounding methods were established com-                    Fort Worth, TX, USA.
pounds known to promote healing (i.e. EGF) were added and the                    Matrix metalloproteinases (MMPs) are responsible for the degra-
responses to these stimuli were monitored. Histology was used to               dation of extracellular matrix components (such as collagens and
determine the amount of re-epithelialization and proliferation,                proteoglycans) and are considered to play an important role in
ELISAs and immunostaining were used to follow protein expression               wound healing. Various studies have confirmed the presence of high
and secretion, and skin permeability assays were used to determine             MMP levels in chronic wound fluid.1 The MMPs that display crucial
percutaneous absorption and barrier properties. This epidermal skin            activity in wounds are interstitial collagenase (MMP-1), gelatinase
wounding model provides a valuable platform to study the cellular              A (MMP-2), gelatinase B (MMP-9), as well as collagenase 3 (MMP-
responses produced in response to actives designed to promote                  13). The Small Intestine Submucosa (SIS) Wound Matrix* is a bioac-
wound healing.                                                                 tive extracellular matrix for wound healing. It contains critical
                                                                               components of the extracellular matrix such as collagens, proteogly-
76                                                                             cans, and glycosaminoglycans. The purpose of this study was to
poly-n-acetylglucoSamine nanoFiBerS From a ma-                                 evaluate the effect on keratinocyte migration produced by the degra-
rine diatom promote Wound healing and angiogen-                                dation products released following exposure of SIS to MMP 1, 2, 9,
eSiS via an aKt1/etS1-dependent pathWay                                        or 13. Active MMPs were mixed into a solution and incubated with
Robin C. Muise-Helmericks, Ph.D.1, Haley Buff1, Aiguo Zhang2,                  SIS wound matrix at 37°C for 24 hours. The materials released from
Juanita Eldridge1, Tiera Liby1, Marina Demcheva, Ph.D.3, Arun                  the MMP degradation were then applied to human keratinocytes
Seth, M.D.4, John N. Vournakis, Ph.D.3.                                        that had been cultured on a gold surface to examine cell migration.
 Medical University of South Carolina, Charleston, SC, USA,                    The test groups also included untreated keratinocyte cultures, ker-
 Sunny Brook Research Institute, University of Toronto, Toronto,               atinocyte cultures that had been exposed to SIS without MMP pre-

                     2009 SympoSium on advanced Wound care and Wound healing Society meeting W31
treatment, and keratinocyte cultures treated with MMPs but no SIS.        80
Keratinocyte migration was dramatically inhibited following treat-        SenSitivity and SpeciFicity oF high reSolution ultra
ment with the MMP solution, while the SIS sample incubated with           Sound (hruS) For the detection oF oSteomyelitiS in
MMP’s demonstrated a much higher migration index. These results           chronic WoundS
indicate that MMPs have an inhibitory effect on keratinocyte migra-       Martin Wendelken, DPM, RN, Lee Markowitz, DPM, Christopher
tion, but the digestion products of SIS treated with MMPs increased       Comfort, MD, Oscar M. Alvarez, PhD.
migration significantly. This data suggests that the SIS material not     Center for Curative and Palliative Wound Care, Bronx, NY, USA.
only functions as a scaffold at the wound site, but also could neutral-     Sixty-eight consecutive chronic wound patients with clinical suspi-
ize MMP’s inhibitory effect on cell migration.                            cion of bone infection were scanned with a high-resolution ultra-
* Oasis® Wound Matrix (Cook Biotech)                                      sound device (Aloka SSD-3500, Aloka Inc, Wallingford, CT)
1. Trengove NJ, Stacey MC, Macauley S, Bennett N, Gibson J, Burslem       equipped with a 7-13 MHz linear array transducer. Wounds were
    F, Murphy G, Schultz G, Wound Rep Reg 1999;7:442-52                   filled with a sterile acoustic amorphous gel, covered with a thin
                                                                          polyurethane film and scanned to obtain both cross-sectional and
78                                                                        longitudinal views. This technique called Wound Mapping* provides
development oF an in vitro model to evaluate the                          a clear illustration of the wounds and surrounding tissues up to a
potential For adherence oF Wound healing dreSS-                           depth of 6 cm. A patient was given a positive diagnosis for os-
ingS                                                                      teomyelitis if the HRUS scan revealed: 1) An echoless zone adjacent
Rachael Clark, PhD1, Michelle Delbono2, Sally-Anne Stephens,              to the cortex of the bone reflecting elevation of the periosteum (sub
BSc2, Omotayo Abioye2, Simon Bayliff2.                                    periosteal abcess); 2) Direct connection of the echoless zone with
 Systagenix Wound Management, Gargave, United Kingdom,                    the wound (fistula formation) and cortical interruption. Following the
 Systagenix Wound Management, Gargrave, United Kingdom.                   ultrsound scan, each patient also had an MRI. A positive MRI was
  introduction Adherence of wound dressing material to the wound          noted if the scan revealed: 1) cortical interruption; 2) sinus tract with
bed has previously been reported, in-vivo, with fibrous dressings         soft tissue mass, and abnormal marrow signal intensity. All images
(Bell & Hart; 2007). Fibres may remain in the wound bed that could        were reviewed retrospectively and separately by 2 blinded review-
negatively affect the inflammatory response, as demonstrated in-          ers. Sensitivity, specificity, positive (PPV) and negative predictive
vivo by giant cell reactions. Adherence of wound dressings can            values (NPV) were calculated for HRUS at 95% CI’s. MRI was con-
occur in clinical practice, possibly as a consequence of changing         sidered the gold standard (100% sensitivity, 75% specificity, 92.9%
wound conditions’ as a wound infection is resolved or healing pro-        PPV, and 100% NPV). Results of HRUS scan showed 97% sensitivity,
gresses.                                                                  92.8% specificity, 95.6 PPV, and 97% NPV). HRUS does require train-
  method A unique quantitative in-vitro model has been developed,         ing but could theoretically be performed by the general practitioner,
based upon the adherence of a wound dressing to a fibrin clot. The        is cost effective (about the same cost as an X-Ray, and 4.6 times less
clot provides an adherent, biological matrix simulating the wound         expensive than an MRI). These results demonstrate that HRUS
bed surface and is applied between two pieces of dressing. The            could be a useful tool for the diagnosis chronic osteomyelitis. Further
force required to remove the dressing material from a fibrin clot is      studies are needed to correlate this rapidly progressing technology
measured.                                                                 to leukocyte bone scans and histopathology.
  A design of experiments approach was employed to investigate all        *Wound Mapping, BioVisual Technologies, LLC Elmwood, NJ
interactions between variables. A number of fibrous (mainly algi-
nate) wound dressings were assessed in conjunction with non-ad-           81
herent wound dressings, including a non-adherent antimicrobial            eFFectiveneSS oF intermittent, pneumatic compreS-
dressing.                                                                 Sion For the treatment oF venouS ulcerS in SuB-
  results The model was optimized for clot size, fibrin and protein       JectS With Secondary lymphedema
concentration and adhesion conditions. The maximum force meas-            Oscar M. Alvarez, PhD, Martin Wendelken, DPM, RN, Gail
ured, gf, to separate the dressing material from the clots for the non-   Chrzanowski, MD, Lee Markowitz, DPM.
adherent wound dressings was 160gf. The force required to                 Center for Curative and Palliative Wound Care, Bronx, NY, USA.
separate the fibrous dressings from the clots was significantly             Fifty-two subjects with secondary lymphedema, chronic venous in-
higher, ranging between 294 -658 gf.                                      sufficiency, and hard to heal lower leg ulceration (>1year old &
  A linear relationship existed between the adherence value for the       >20cm2 surface area) were treated with either intermittent, gradient,
dressing and the percent open area, dependent on the diameter of          pneumatic compression (IPC* n=27) plus standard compression
the hole in the non-adherent layer.                                       therapy or compression therapy alone (control). Compression ther-
  conclusion Decreasing the potential adherence of dressings and          apy consisted of a non-adherent primary wound dressing plus a 4
limiting the number of fibres that remain at the wound surface, may       layer compression bandage (4-LB** n=25). The mean age and size
positively influence the wound healing process. This model distin-        of the ulcers were 1.4 years and 31cm2 respectively and did not dif-
guishes between non-adherent wound dressings and dressings that           fer significantly between groups. IPC was performed using a 4-
have a potential to adhere to a wound bed. This in-vitro model has        chamber pneumatic leg sleeve and gradient, sequential, pump. All
the potential to be used as a replacement to current in-vivo models.      pumps were calibrated to a pressure setting of 50 mmHg on each
                                                                          subject and treatments were for 1hour twice daily. Evaluations were
                                                                          performed weekly to measure edema, local pain, degree of wound
                                                                          granulation, and wound healing (incidence of complete closure and

W32 2009 SympoSium on advanced Wound care and Wound healing Society meeting
rate of healing from wound surface area measurements). The me-              83
dian time to wound closure by 9 months was 141 days for the IPC-            Studying the healing eFFicacy oF ho/03/03 utiliZing
treated group and 211 days for the control group (P= 0.031). The rate       pictZar® cdm vS. visitraktm Wound meaSurement SyS-
of healing was 0.8±0.4 mm/day for the control group and 2.1±0.8             tem methodS.
mm/day for the group treated with IPC (P<0.05). When compared               Moshe Ben Hamo, Eli Braude, Ofra Levy-Hacham, Liora Braiman-
to subjects treated with standard care, the group treated with IPC          Wiksman, Tamar Tennenbaum.
reported less pain at each evaluation point for the first 6 weeks of        HealOr Ltd., Rechovot, Israel.
the trial. At week 2 and 3 the visual analog pain scores were signifi-        introduction: Accumulative evidence show that wound area meas-
cantly lower for the IPC-treated group (P<0.05). These results sug-         urements at 4 weeks can be prognostic of complete wound closure.
gest that IPC is a valuable adjunct to compression therapy in the           The acetate tracing method VisitrakTM (Smith&Nephew) is com-
management of large or painful venous ulcers.                               monly used for wound measurement. Recently, a new method was
* Sequential Circulator Model 2004 Bio Compression Systems, Inc.,           introduced, PictZar® (Medline Industries, Inc) a Calibrating Digital
   Moonachie NJ                                                             Measurement Planimetry Software based on photo-documenting
** Profore Compression Bandage System Smith and Nephew, Inc., Largo         analysis, for wound area calculation.
   FL                                                                         This study compared both methods on diabetic foot ulcers (DFUs).
                                                                              methodology: HealOr performed a wound healing study in Israel
82                                                                          on DFU patients assessing safety and preliminary efficacy of a new
can BiomarKerS Be uSed to predict clinical eFFi-                            topical drug, HO/03/03. During the study, each wound was sub-
cacy?                                                                       jected to daily measurements utilizing PictZar® CDM and VisitrakTM.
Breda Cullen, PhD1, Alicia Essler, BSc1, Katherine Wallenfang-                results: Both techniques demonstrated that HO/03/03 is a promi-
Sohle, MD2, Robert Stadler, MD2.                                            nent drug for healing of chronic DFUs. The results demonstrated
 Systagenix Wound Management, North Yorkshire, United King-                 that 10/15 treated wounds healed within 14 weeks of a 20 week trial.
dom, 2Minden Hospital, Minden, Germany.                                     Furthermore, 9/10 of the wounds which closed, demonstrated a de-
  The incidence and prevalence of chronic wounds is increasing and          crease in wound size of more than 50% at 4 weeks where two of the
becoming a bigger burden to health care budgets. While this is in           healed wounds completely healed at 4 weeks. Strong correlation
part due to a growing elderly population and an increase in obesity,        was observed in wound area calculations, performed by both tech-
it is also a reflection on the variable clinical success of current prod-   niques. VisitrakTM yielded wider and more continuous graphs, while
ucts.                                                                       PictZar® graphs were characterized by accuracy and high resolu-
  One of the greatest challenges in wound care is determining which         tion of the wound dimension corroborated by statistical analysis. Vis-
product to use and when. Treatment choice is typically based on vi-         itrakTM was subjected to inaccuracies, associated with manual
sual assessment with minimum analysis of the underlying patho-              on-sight measurements. PictZar® photo-documentation which uses
physiology; this is highly subjective, and decision relies on trial and     a computerized measurement system could be stored, retrieved and
error. Due to the high cost of failure more expensive advanced ther-        analyzed utilizing objective parameters independently by different
apies have not been widely adopted; instead they are reserved for           personnel. Furthermore, at graph margins, PictZar® was shown to
the worst-case wounds. However, if we could predict which patients          be more sensitive as compared to VisitrakTM . VisitrakTM was insen-
would respond to a therapy, the true benefit of advanced therapies          sitive below 0.3 cm2, whereas PictZar® identified wound opening
would be realized.                                                          of 0.1-0.2 cm2. In conclusion: Our results confirm previous studies
  In this study, we have correlated the clinical efficacy of a Colla-       suggesting 4 weeks time point as a predictor of wound healing and
gen/ORC product with biomarkers present in the wound fluid.                 suggests PictZar® to be more sensitive than VisitrakTM in analyzing
Wounds were monitored bi-weekly for clinical progress and wound             full closure of wounds during treatment.
fluids collected from patients throughout their 12-week treatment,
to facilitate analysis using standard in vitro methods. Our results in-     84
dicate that wounds, which responded to Collagen/ORC, also exhib-            the treatment oF patientS With Stage iii and iv preS-
ited a decrease in protease activity and an increase in the ratio of        Sure ulcerS With thymoSin Beta 4 (tβ4); a SaFety and
inhibitor to enzyme, suggesting better protease regulation post ther-       eFFicacy Study
apy. This clinical evidence validates our previous in vitro and ex vivo     Terry A. Treadwell, MD1, Luigi S. Girardi, MD2, Yuxin Zhang, PhD2,
studies, showing that Collagen/ORC binds and inactivates pro-               David R. Crockford2, Linda A. Martin2.
teases. We believe it is necessary to provide both clinically relevant      1
                                                                             Institute for Advanced Wound Care, Montgomery, AL, USA, 2Re-
data and multiple levels of scientific and clinical evidence to support     geneRx Biopharmaceuticals Inc, Bethesda, MD, USA.
product claims.                                                               introduction: Fourteen US clinical sites participated in a Phase II
  In addition, biomarker analysis pre-therapy suggests they could be        pressure ulcer study that evaluated the safety, tolerability, wound
used as predictors of efficacy for Collagen/ORC products. Adopt-            healing effectiveness, and potential antibody response of 3 escalat-
ing this personalized approach would allow the caregiver to imme-           ing Tα4 gel concentrations (0.01%, 0.02%, and 0.1%) applied topically
diately determine when product use was appropriate to facilitate            once daily (qd) for up to 84 consecutive days.
healing; this would have clear economic benefits and improved clin-           methods: Seventy-two patients having Pressure Ulcers with a sur-
ical outcomes.                                                              face area between 5 and 70 cm² were randomized using a Tα4:
                                                                            placebo ratio of 3:1. Dose-escalation occurred by cohort and was
                                                                            preceded by a blinded review of safety data by a Core Committee

                    2009 SympoSium on advanced Wound care and Wound healing Society meeting W33
comprised of the Sponsor, Primary Investigator, Biostatistician, and       86
Medical Monitor after all cohort subjects had undergone 28 days of         inhiBition oF multidrug-reSiStant acinetoBacter
treatment and safety evaluations. Cohort 1 (0.01% Tα4 gel or placebo)      Baumannii By non-viral eXpreSSion oF hcap-18 in a
contained 24 subjects (22 male, 2 female) ranging from 22 to 83 in         Bioengineered human SKin tiSSue
age. A total of 3 Serious Adverse Events (SAEs) were reported; none        John Centanni1, Christina Thomas-Virnig, Ph.D1, Colette John-
deemed related to study drug. Cohort 2 (0.02% Tα4 gel or placebo)          ston1, Li-Ke He, M.D.2, Sandy Schlosser3, Kelly Van Winkle1, Ruib-
contained 24 subjects (16 male, 8 female) ranging from 19 to 85 in         ing Chen3, Angela Gibson3, Andrea Szilagyi2, Lingjun Li, Ph.D3,
age. A total of 4 SAEs were reported; none deemed related to study         Shankar Ravi, Ph.D2, B. Lynn Allen-Hoffmann, Ph.D3.
drug. Cohort 3 (0.1% Tα4 gel or placebo) contained 24 subjects (17         1
                                                                            Stratatech Corporation, Madison, WI, USA, 2Loyola University
male, 7 female) ranging from 25 to 83 in age. The last subject in Co-      Medical Center, Maywood, IL, USA, 3University of Wisconsin,
hort 3 will complete study participation in November 2008.                 Madison, WI, USA.
  results: After database lock, the Sponsor will report all unblinded        purpose: Bacterial contamination is the most common reason for
safety data, efficacy measures, and pharmacokinetic data.                  impairment of wound healing. During the healing response, ker-
  conclusions: The acceptable safety profile in 72 patients enabled        atinocytes produce host defense peptides (HDPs) that have antimi-
dose escalation per study design. Unblinded data will provide con-         crobial activity against a diverse set of pathogens. The goal of this
firmation of the blinded safety results and new evidence of efficacy.      study is to genetically engineer a human skin tissue to express the
Bibliography: [1]                                                          human cathelicidin HDP (ExpressGraft™Enhance) that could ulti-
A Randomized, Double-Blind, Placebo-Controlled, Dose-Response Study        mately be applied to burns and ulcers to counteract bacterial con-
   of the Safety and Efficacy of Thymosin Beta 4 in the Treatment of Pa-   tamination and prevent infection.
   tients with Pressure Ulcers                                               methods: A non-viral vector containing the human cathelicidin
                                                                           (CAMP) cDNA was used to express cathelicidin (hCAP-18 and the
85                                                                         mature peptide LL-37) in the NIKS® keratinocyte cell line. Clonal iso-
WoundS meaSured uSing photo-digital planimetry                             lates of stably-transfected cells were isolated. To assess antimicro-
SoFtWare (pdpS): validation and inter/intra-rater                          bial activity in vitro, topical extracts from ExpressGraft™Enhance
reliaBility When compared to Wound tracingS                                tissue were incubated with medium containing Staphylococcus
Will Berg, Martin Wendelken, DPM, RN, Lee Markowitz, DPM,                  carnosus. in vivo antimicrobial studies evaluated ExpressGraft™En-
Christopher Comfort, MD, Oscar M. Alvarez, PhD.                            hance in a murine burn infection model where full-thickness scald
Center for Curative and Palliative Wound Care, Bronx, NY, USA.             burns were inoculated with A. baumannii. NIKS® and Express-
  The gold standard for measuring wounds has been tracing and              Graft™Enhance tissues were grafted on these wounds after eschar
planimetry (tracing the perimeter of the wound on transparent ac-          was removed. Samples were harvested at 72 hrs for quantitative
etate with a fine-tip marker, then calculating the surface area by         bacteriological cultures.
counting grids manually, or with a digitized pad). A wound measure-          results: ExpressGraft™Enhance expresses approximately 140-
ment method using PDPS* was compared to standard tracings                  fold more hCAP-18/LL-37 than unmodified NIKS® tissue and pos-
(measured manually and with a digitized pad**) in more than 200            sesses key histological features of normal epidermis. Peptides
wounds of all shapes and sizes. Intra-rater and inter-rater reliability    extracted from the surface of ExpressGraft™Enhance inhibited
of wound measurements obtained from tracings and from PDPS                 growth of S. carnosus by over 78% as compared to unmodified
were studied and compared. Rater variability was directly related to       NIKS® tissue, confirming the antimicrobial properties of the Ex-
wound size and shape when wounds were traced in the traditional            pressGraft™Enhance. Moreover, in an in vivo infected burn wound
manner. In small (<4cm2), regularly shaped (round or oval) wounds          model, ExpressGraft™Enhance results in a two log reduction in a
both accuracy and rater reliability was 98±2% and 95±3.5% respec-          clinical isolate of multidrug-resistant A. baumannii.
tively with tracings or PDPS. However, in larger and/or irregularly          conclusions: Non-viral genetic engineering of ExpressGraft™ En-
shaped wounds, or wounds with epithelial islands, significantly            hance alleviates concerns of safety and heterogeneity associated
larger standard errors were noted with traditional tracings (17% for       with viral transfection. The application of ExpressGraft™Enhance
tracings and 6% for PDPS, P=0.023). The mean inter-rater reliability       to cutaneous wounds will not only provide temporary epithelial cov-
score was 80% for traditional measurements, 84% for digitized pad          erage and growth factors similar to currently available skin substitute
planimetry and 94% for PDPS P<0.05). In addition to the obvious ad-        products, but our studies provide compelling evidence that Express-
vantages (handling the tracing, and difficulty seeing the wound            Graft™Enhance is able to combat multidrug-resistant nosocomial
edges clearly), planimetry of digital photographs allows for a closer      strains that are of great concern to the medical community.
examination of the wound, and better visibility of advancing epithe-
lium. It also permits quantitation of other wound assessment param-        87
eters such as granulation, nonviable tissue, erythema and                  an in vtro model For teSting topical antimicroBial
maceration. All 5 examiners felt that PDPS was more accurate, and          FormulationS againSt Bacterial BioFilmS
more objective than traditional tracings. PDPS also resulted in better     Catherine Van Der Kar, Eric Roche.
correlation within and between examiners. With PDPS, wounds may            Healthpoint Ltd., Fort Worth, TX, USA.
be archived and studied serially, plus wound measurement data can            A growing body of evidence indicates that bacterial biofilms are fre-
be exported to the chart or other software programs such as Stat-          quently present in chronic wounds. This is cause for significant clin-
Soft, SPSS, Word or Excel for tabulation and analysis.                     ical concern given the reduced efficacy of antimicrobial agents
*PictZarTM-CDM, BioVisual Technologies, LLC, Engelwood, NJ)
                                                                           against biofilm bacteria. The scientific community has developed a
** VisitrakTM Smith and Nephew Inc, Largo FL

W34 2009 SympoSium on advanced Wound care and Wound healing Society meeting
variety of in vitro models to test antimicrobial efficacy against          89
biofilms; however, these models are designed to test active mole-          a porcine model oF mrSa inFection in Full thicK-
cules in solution against bacteria growing on artificial surfaces. Here    neSS WoundS With reproduciBle delayed healing
we describe the adaptation of an in vitro model to enable testing of       Paul Renick, MS1, Eric Roche, PhD1, Dale Telgenhoff, PhD1, Egee-
topical formulations of antimicrobials against bacteria growing in a       nee Daniels, DVM2, Lawton Seal, PhD1, Duncan Aust, PhD1.
collagen-based matrix. A piece of collagen-based matrix was placed         1
                                                                            Healthpoint Ltd., Fort Worth, TX, USA, 2University of Texas Health
on a membrane resting on a blood agar media surface. The collagen          Science Center, Fort Worth, TX, USA.
matrix was inoculated with the test bacteria, and the assembly incu-         Wound infection is a known factor in the delayed healing of many
bated to foster bacterial growth. At desired time points, treatment        chronic human wounds, and pigs are a preferred model system for
was initiated by applying a topical formulation to moistened gauze         acute wound healing because of the similarity of human and porcine
and placing the formulation in direct contact with the collagen matrix.    skin. Here we describe a model for MRSA infection of full thickness
When treatment with commonly used antimicrobial cream products             porcine wounds and the delay in wound healing that results from the
was initiated shortly after bacterial inoculation, some of the creams      infection. Repeated inoculation of full thickness wounds led to a
exhibited substantial efficacy. In contrast, when the bacteria were        MRSA bioburden in the wound tissue that was consistently above
allowed to form a visible community in the matrix over 24 hours, the       the 105 cfu / g threshold guideline for human wounds. Measure-
antimicrobial creams tested had little effect on the bacterial load.       ments of wound size and wound closure over time demonstrated
These results support the biofilm character of this model and em-          that wounds receiving mock inoculations closed more rapidly than
phasize the need for new topical antimicrobial products with greater       wounds inoculated with MRSA. The microbial load and delayed heal-
efficacy against biofilms. in vitro and in vivo models for biofilm wound   ing were reproduced across several studies and a number of ani-
infection will provide crucial guidance for the development of novel       mals. Treatment of wounds with silver dressings in one study did not
treatments.                                                                significantly affect the tissue counts of MRSA or improve healing.
                                                                           This infected, delayed-healing model offers a valuable tool for testing
88                                                                         both marketed and novel treatments for their effects on wound
molecular diagnoSticS to improve therapy choice                            bioburden and healing.
and enhance Wound care
Scot E. Dowd, Ph.D.1, Randall D. Wolcott, MD2.                             90
 Research and Testing Laboratory, Lubbock, TX, USA, 2Southwest             development oF a Field-ready antimicroBial Wound
Regional Wound Care Center, Lubbock, TX, USA.                              dreSSing For the treatment oF cutaneouS veSicant
  Molecular diagnosis of wound infections presents a relatively new        WoundS
field of interest in wound care, which we have already shown to have       Barry Steiglitz, Ph.D., Kenneth Gratz, Ph.D., Priya Nataraj, B.S., Co-
remarkable utility clinically. The use of culture based diagnostics se-    lette Johnston, B.S., Kelly Van Winkle, B.S., Nicholas Simon, B.S.,
lects for only those organism which are able to be cultivated readily      Sara Pirnstill, B.S., Kristi Schaeve, B.S., Sarah Sisson, B.S., Lynn
in general laboratory media. Examples of easily cultivated organisms       Allen-Hoffmann, Ph.D..
include Staphylococcus, Serratia, and Pseudomonas spp. Culture             Stratatech Corporation, Madison, WI, USA.
based detection takes several days to provide results and often select       purpose: Current conflicts in Iraq and Afghanistan have seen the
for minor populations in wounds because these populations are read-        number of field burns reach new levels. The susceptibility of these
ily cultivated. A new chronic wound diagnostic panel developed by          wounds to infection and the increasing incidence of multi-drug resist-
Research and Testing Laboratory and put into practice by Southwest         ant clinical isolates of bacteria highlight the need for novel ap-
Regional PCR and the Southwest Regional Wound Care Center for              proaches to combat infection. We are developing EpiReadyDefense,
diagnosing wound pathogens was evaluated with comparison to cul-           a tissue-engineered antimicrobial therapeutic designed to promote
ture based diagnostics, and a gold standard sequencing approach.           healing by providing barrier function and delivering antimicrobial pep-
A number of cases in a cohort are presented, which highlight the util-     tides to wound beds. We have developed processes that render skin
ity of this new rapid molecular diagnostics approach in improving          substitute tissues nonviable, thus prolonging ambient temperature
wound care. The benefits include reduced cost of medication, im-           storage capacity while preserving wound coverage capability and fa-
proved rate of healing, and improvement in the morale of both clini-       cilitating delivery of antimicrobial peptides to cutaneous vesicant or
cians and patients. Example cases in which empirically indicated and       thermal injuries sustained in the field.
prescribed antibiotics were counter indicated by molecular methods           methods: The clinically tested NIKS® keratinocyte line was en-
allowing targeted therapies to be applied are highlighted including        gineered by stable transfection with a non-viral vector to overex-
molecular detection of Candida albicans, MRSA, Pseudomonas,                press broad spectrum host defense peptides. Fully-stratified skin
VRE, and HSV1. Finally the utilization of primary antibiotic marker de-    substitutes were constructed by culturing these cells on a fibrob-
tection and how molecular methods can help guide sensitivity testing       last-embedded dermal substrate. Skin substitutes were termi-
and guide choice of onsite therapy are provided. Results show that         nally processed by drying or gamma irradiation with 65Co in
culture based diagnostics have a low success rate in identifying the       order to eliminate cellular viability, and evaluated for histology,
major populations of bacteria in wounds and are very slow (4 days)         biomechanical properties, metabolic activity, and cell outgrowth
while molecular methods are fast (< 3 hrs onsite and < 24 hrs point of     from tissue explants. Topical extracts isolated from skin substi-
service) and much more effective and cost effective in guiding ther-       tutes were incubated with cultures of Staphyloccus carnosus and
apy. Sequencing based and molecular methods are the future and             antimicrobial activity was determined by quantifying residual bac-
next generation of diagnostics for chronic wounds.                         terial colony forming units.

                    2009 SympoSium on advanced Wound care and Wound healing Society meeting W35
  results: Terminally processed skin substitutes retained normal tis-       92
sue architecture and exhibited ultimate tensile strength values indis-      a SKin SuBStitute tiSSue Bioengineered uSing a non-
tinguishable from those of freshly-prepared skin substitutes.               viral vector to eXpreSS timp-1 inhiBitS metallopro-
Processing of skin substitutes eliminated fibroblast cell outgrowth         teinaSe activity
from explants and reduced metabolic activity by greater than 90%.           Christina Thomas-Virnig, Ph.D, Cathy Rasmussen, Nick Simon,
Extracts from terminally processed skin substitutes reduced bacte-          Sarah Sisson, Mary Lokuta, Ph.D, B. Lynn Allen-Hoffmann, Ph.D.
rial growth by 61% relative to control cultures of S. carnosus.             Stratatech Corporation, Madison, WI, USA.
  conclusions: These proof of principle studies establish that antimi-        purpose: Successful wound closure requires balanced matrix met-
crobial skin substitutes can be processed in order to preserve tissue       alloproteinase (MMP) and tissue inhibitor of metalloproteinase
architecture, physical properties, and antimicrobial activities, sup-       (TIMP) activity for proper granulation tissue formation and ker-
porting the further development of a complex bioactive wound                atinocyte growth and migration. Chronic cutaneous wounds exhibit
dressing for treatment of cutaneous injuries.                               a highly proteolytic environment due to an increase in MMP activity
                                                                            and a corresponding decrease in TIMP activity inhibiting the healing
91                                                                          process. The goal of this study is to develop a genetically modified
17β-eStradiol inJectionS accelerate Wound healing                           therapeutic skin substitute with a non-viral vector that reduces the
and eXhiBit a narroW doSe reSponSe range in Both                            proteolytic imbalance of chronic wounds by expressing elevated lev-
animalS and man                                                             els of TIMP-1.
Nick Occleston, BSc (Hons), MPhil, PhD, Neil French, BSc                      methods: A cDNA encoding TIMP-1 was cloned into non-viral ex-
(Hons), MSc, PhD, Abdul Sattar, BSc(Hons), MSc, PhD., Adam                  pression vectors. These vectors were stably transfected into NIKS®
Boanas, BSc, Tracey Mason, BSc (Hons), PhD, David Fairlamb,                 keratinocytes. Fully-stratified skin substitutes were prepared from
BSc (Hons), Mark Cooper, BSc(Hons), MSc, Jim Bush, MBChB,                   the modified keratinocytes and assessed for histology and protein
MRCS (Ed), Sharon O'Kane, BSc (Hons), PhD, Mark W. J. Fergu-                expression. Tissue was evaluated in vitro for inhibition of MMP-2 pro-
son, BSc (Hons), BDS, PhD, DMedSci..                                        teolytic activity using an EnzChek Collagenase Kit (Invitrogen, Carls-
Renovo, Manchester, United Kingdom.                                         bad, CA).
  Pre-clinical studies evaluating the effects of intradermal adminis-         results: Clonal isolates of all stably transfected NIKS® cells
tration of an aqueous formulation of 17α-estradiol on the acceleration      (NIKSTIMP-1) form fully-stratified skin substitute tissue (Express-
of healing were conducted using punch biopsies in Sprague Dawley            Graft™Shield) that secretes as much as 18-fold higher TIMP-1 than
female (ovariectomised) and male rats and split-thickness skin graft        unmodified tissue. Indirect immunofluorescence of the tissue
donor sites in male Large White Yorkshire pigs. Dose response               formed from the highest expressing NIKSTIMP-1 clone shows that
(0.001-10αg/100αL) and dose frequency (1, 2 and 3 administrations)          TIMP-1 is expressed throughout the epidermis with the highest con-
were investigated using histological measures of wound re-epithe-           centration in the granular layer. Conditioned medium from Express-
lialisation as the study endpoint. Translation of these data to man         Graft™Shield inhibits MMP-2 activity by as much as 71% compared
were studied in a double-blind, within-subject, placebo controlled          to unmodified tissue in an in vitro fluorescent gelatin cleavage assay.
clinical trial (a total of 44 subjects) in both male (aged 18-85) and fe-     conclusions: NIKSTIMP-1 cells produce fully-stratified skin tissue
male (post-menopausal) volunteers. This used a punch biopsy                 indicating that keratinocyte terminal differentiation progresses nor-
wound evaluating single administrations of 17α-estradiol at doses of        mally in overexpressing cells. The TIMP-1 found in the conditioned
0.02-0.4αg/100αL, with histological measures of wound re-epithelial-        media exhibits proper secretion from ExpressGraft™Shield and
isation as the primary healing endpoint.                                    demonstrates functionality against a chronic wound metallopro-
  Preclinical studies in rats and pigs demonstrated that single admin-      teinase. Notably, the non-viral genetic engineering of this skin sub-
istrations of 17α-estradiol significantly accelerated the healing of        stitute tissue alleviates many safety concerns as well as the
wounds with a narrow efficacious dose range between 0.01 and                heterogeneity associated with viral transfection. Ultimately, we hope
0.4αg/100αL. Lower doses had no beneficial effect and higher doses          to translate this technology into the clinic to combat the highly pro-
(>0.4αg/100αL) impaired healing. Dosing frequency studies demon-            teolytic environment of chronic cutaneous wounds.
strated that single applications were as efficacious as repeat dosing
(2 or 3 times). The human clinical trial demonstrated a statistically       93
significant improvement (p = 0.027) in healing at the 0.1αg/100αL           BioFilm Formation inhiBition By Sanguitec® engi-
dose, accelerated healing at the 0.2αg/100αL dose and reduced ep-           neered dreSSing SyStemS in a chronic Wound
ithelial re-growth at the 0.4αg/100αL dose. Intradermal administration      BioFilm model
of 17α-estradiol was safe and well tolerated.                               John P. Kennedy, Ph.D.1, Curtis E. Jones, II, Ph.D.1, Scot E. Dowd,
  These data demonstrate that a single intradermal administration           Ph.D.2
of 17α-estradiol accelerates wound healing and that the efficacy of         1
                                                                             South University, Savannah, GA, USA, 2Research & Testing Lab-
this therapeutic approach resides within a narrow dose range. These         oratories, Medical Biofilm Research Institute, Lubbock, TX, USA.
data also demonstrate that high doses can impair healing. The dose            purpose: The present study was undertaken to delineate the ability
responses observed provide evidence of a clear translation from             of two prototype Sanguitec® Lipogel™ Engineered Dressing Sys-
preclinical into clinical models.                                           tems to inhibit the progression of biofilm formation utilizing the Lub-
                                                                            bock Chronic Wound Biofilm (LCWB) model.
                                                                              methods: Aliquots of the prototype Sanguitec® Lipogel™ dressing
                                                                            formulations were dissolved, diluted with media and inoculated in

W36 2009 SympoSium on advanced Wound care and Wound healing Society meeting
triplicate with model species (Ps. aeruginosa PAO1, E. faecalis V583,           6]>200pg/ml, resulting in hyper-proliferation of fibroblasts and a pro-
and S. aureus Mu50). Controls were generated using the same prep                migratory environment for keratinocytes. Skewing the cell ratio in ei-
methods. Treatment and control systems were monitored for biofilm               ther direction resulted in aberrant cytokine profiles, disrupting
growth on polypropylene substrates while incubated under shaking                reciprocal communications between the two cell types, and ulti-
at 370C and 140 RPM for 24 hr. All systems were removed following               mately resulting in poor epidermal development. Our studies have
incubation and planktonic bacteria were separated from residual                 provided greater insight into the critical limits of preserving the sig-
biofilm phenotypes. Biofilm residuals were dried in at 800C for 48              naling dynamics between the cellular compartments during BLSS
hr and the dry weight of biofilm formed was determined. The con-                manufacturing. This may have wide implications for the development
centrations of specific bacterial species within the biofilms were de-          of wound healing therapies utilizing signaling molecules.
termined using PCR methods.
  results: Both Sanguitec® Lipogel™ prototype formulations com-                 95
pletely inhibited biofilm formation at concentrations of 0.1% or                the eFFect oF metal ionS on enZymatic activity oF
greater. Further, even at concentrations as low as 0.05% and 0.01%              cloStridium collagenaSe
the prototype Lipogel™ formulations inhibited biofilm formation an              Aleksa V. Jovanovic, Ph.D., Ryan Ermis, B.S., Lei Shi, Ph.D..
average of 71.3% (Form A- 58.7%, Form B- 83.8%) and 49.0% (Form                 Healthpoint Ltd., Fort Worth, TX, USA.
A- 40.6%, Form B- 57.5%) respectively. Conclusions: These initial pro-            The healing of wounds is a complex process, which is often further
totypes of Sanguitec® Lipogels™ offer effective biofilm inhibition              complicated by the presence of non-viable, necrotic tissue in the
in the in vitro LCWB model in concentrations as low as 0.1%. In ad-             wound area. Debridement is the process of removing the non-viable
dition, these Sanguitec® Lipogel™ systems showed little or no                   tissue from a wound to prevent or diminish infection and to promote
species selectivity with respect to inhibition as is seen typically in          healing. Proteolytic debriding enzymes, such as collagenase, have
other biofilm agents. While the potency demonstrated by these pro-              been used for the wound debridement for decades. Clostridium col-
totype Lipogels™ in the LCW Biofilm model was notably dramatic                  lagenase is a metalloproteinase, which can specifically attack colla-
(as low as 0.1%), these finding are of particular promising signifi-            gen debris in necrotic tissues. The authors have found that certain
cance considering that these systems may be utilized at concentra-              metal ions, in the form of water-soluble salts can enhance the enzy-
tions 100 fold greater (undiluted) in clinical practice.                        matic activity of C. collagenase in vitro. The following metal ions were
                                                                                studied for their effects on collagenase activity: barium (Ba++), zinc
94                                                                              (Zn++), copper (Cu++), manganese (Mn++), cobalt (Co++), sodium
the impact oF cell ratio on croSS-talK BetWeen Fi-                              (Na+), potassium (K+), calcium (Ca++), nickel (Ni++), and magne-
BroBlaStS and KeratinocyteS during the produc-                                  sium (Mg++). Among these ions, only Na+, K+, and Mg++ enhanced
tion oF human Bilayered living SKin SuBStitute                                  the enzymatic activity of collagenase. Ca++ displayed both enhance-
(BlSS)                                                                          ment and inhibitory effects depending upon the concentration. The
Dolores Baksh, PhD, Vincent Ronfard, PhD.                                       other metal ions tested either inhibited the activity or had no effect
Organogenesis Inc., Canton, MA, USA.                                            on the activity. The only difference observed between the mono- and
  The successful maturation and function of BLSS is dependent on                divalent anions in salts with a common cation was that the divalent
precisely orchestrated cross-talk between fibroblasts and ker-                  anions could increase the activity of collagenase more than the
atinocytes. The fibroblasts provide the necessary growth factors and            monovalent anions. This is the first time to find that Na+, K+, and
extracellular matrix signals that support keratinocyte growth, termi-           Mg++ could enhance the activity of C. collagenase. These results
nal differentiation and survival. Keratinocytes provide positive feed-          could help clinicians better understand how C. collagenase can be
back to the fibroblasts to continue their appropriate differentiation           used most effectively in combination with therapies containing metal
programs. This study varied the Fibroblast:Keratinocyte ratio (F:K)             salts.
to evaluate the impact on signaling cytokine levels and its correlation
to the development of functional BLSS produced by our current                   96
manufacturing process for Apligraf®. This process involves a der-               in vivo aSSeSSment oF the gelSpray™ hydrogel
mal matrix containing fibroblasts embedded in collagen, which is                Wound dreSSing
then seeded with keratinocytes.                                                 Sameer Shums, MBA, John Jackson, BS, Thomas Hirt, PhD.
  The number of fibroblasts impacts the keratinocytes in several crit-          BioCure, Inc., Norcross, GA, USA.
ical ways including their attachment, migration, proliferation and dif-           BioCure’s GelSpray™ and antimicrobial GelSpray™ dressings
ferentiation. Inability to achieve any one of these biological functions        were evaluated in three pilot porcine studies, including a 48-hour
will fail to produce a fully matured epidermis. Examination of 6 dif-           barrier evaluation against infection, a 2nd degree burn healing and
ferent F:K ratios (0.075:1, 0.15:1, 0.3:1, 0.6:1, 1.2:1, 2.4:1) revealed that   a partial thickness wound healing study both conducted over 14
there is an optimal relationship between the two cell types to pro-             days. Wounds for the barrier and partial thickness studies were cre-
duce the proper structures and functions of skin.                               ated using an electrokeratome while 2nd degree burns were created
  A low F:K ratio (<0.3:1) during the dermal development stage re-              with heated brass rods. The barrier study required a suspension of
sulted in abnormal cytokine patterns. Essential growth factors critical         Ps. Aeruginosa (ATCC 27312) to be applied external to the dress-
for keratinocyte differentiation were down regulated (e.g,                      ing. The dressings were removed to determine the presence of Ps.
VEGFA<180pg/ml ). In addition, fewer keratinocyte stem cells were               aeruginosa by wash extract of the wound bed. In both wound heal-
maintained, as shown by <7% BrDU incorporation and <12% colony                  ing studies, the wounds from each treatment group and the sur-
forming efficiency. In contrast, a high F:K ratio (>0.3:1) caused [IL-          rounding skin were excised using an electrokeratome. The

                     2009 SympoSium on advanced Wound care and Wound healing Society meeting W37
samples were processed to separate the dermis from the epider-           presence on the same surface as FN-CBD. These findings have im-
mis, allowing for visualization of macroscopic defects or lack of        portant therapeutic implications for FN-deficient wounds, such as
complete epithelialization.                                              burns and chronic ulcers.
  results: The GelSpray™ product demonstrated a 2.5 log reduction
in Ps. Aeruginosa within the barrier studies, while the antimicrobial    98
GelSpray™ showed a 6.5 log reduction as compared to the gauze            compariSon oF Scarring in mice, ratS, pigS and man
control. Epithelialization in the burn study began on Day 10 after       ShoWS comparaBility oF gene eXpreSSion proFileS
wounding and the antimicrobial GelSpray™ achieved 80% epithe-            Anthony Metcalfe, BSc (Hons), PhD, Neil French, BSc (Hons),
lialization while the GelSpray™ dressing accounted for 20%. Air ex-      MSc, PhD, Nick Burgoyne, BSc (Hons), MRes, PhD, Hugh
posed (untreated) wounds achieved 60% epithelialization on Day           Laverty, BSc (Hons), PhD, Abdul Sattar, BSc (Hons), MSc, PhD,
10. The percent epithelialization continued to increase and the an-      Adam Boanas, BSc, Mark W. J. Ferguson, BSc (Hons), BDS,
timicrobial GelSpray™ had complete epithelialization on Day 11           PhD, DMedSci, Sharon O'Kane, BSc (Hons), PhD, Nick Occle-
while, the GelSpray™ and the air exposed control demonstrated            ston, BSc (Hons), MPhil, PhD.
complete epithelialization on Day 12. Both the GelSpray™ and an-         Renovo, Manchester, United Kingdom.
timicrobial GelSpray™ accelerated wound healing in the partial             Several preclinical models have been used to evaluate potential
thickness study at an equal rate with complete epithelialization by      scar reduction therapies, although the utility of these models for
Day 8 while the air exposed, untreated wounds healed completely          comparison to the processes occurring in, and translation to, man
by Day 9. BioCure’s GelSpray™ dressings are an ideal barrier to in-      have yet to be extensively characterised.
fection, creating a moist wound environment to promote healing and         The molecular and histological characteristics of healing and scar-
serves as an effective delivery vehicle for active ingredients such as   ring following incisional wounding in preclinical models including
antimicrobial agents.                                                    mouse, rat and pig were compared to those in man via longitudinal
                                                                         study designs. Following wounding, in both the preclinical and
97                                                                       human (volunteer) model systems, sites were macroscopically as-
preSence oF Both a FiBronectin groWth Factor-                            sessed for healing and scarring and samples taken for gene expres-
Binding domain and the central cell-Binding do-                          sion and histological analysis at a number of time points out to scar
main on a SurFace iS reQuired For FiBroBlaSt                             maturation (>70 days in mouse and rat; and 6 and 12 months in pig
Survival and groWth                                                      and humans respectively). Samples were evaluated histologically
Richard A. Clark, MD1, Fubao Lin, PhD1, Marcia G. Tonnesen,              using standard stains and immunocytochemistry and for gene ex-
MD2.                                                                     pression analysis using Affymetrix gene chips (≥7 timepoints and
 Stony Brook University, Stony Brook, NY, USA, 2Northport                multiple replicates in each species).
VAMC, Northport, NY, USA.                                                  Macroscopic assessments in preclinical models demonstrated that
  During normal wound healing, fibroblast ingrowth is the rate-limit-    both mice and pigs heal with very good scars. Rats heal with the
ing step for granulation tissue formation and appears to require fi-     most pronounced scars which was supported by histological as-
bronectin (FN), which is absent in chronic wounds and burns. The         sessment. Global comparison of gene expression of the healing and
failure of chronic wounds to heal and the progression of injury in       scarring responses by Principal Component Analysis between rats
burns are both associated with fibroblast apoptosis. We have found       and humans demonstrated that although the timescale of events be-
that FN contains 4 growth factor-binding (FN-GFB) sites for PDGF         tween species are different, the overall global gene expression pat-
that are required for fibroblast survival and proliferation. To deter-   terns are highly correlated. Analysis and comparison of specific
mine the mechanisms by which fibroblasts fail to survive and grow        gene changes involved the inflammatory, granulation tissue deposi-
in the absence of FN-GFB sites, mouse FN-null fibroblasts were uti-      tion and remodelling phases of the healing and scarring response
lized. Null- cells cultured in DMEM on recombinant RGD-containing        using clustering techniques demonstrated significant comparability
FN cell-binding domains (FN-CBD), but not any FN-GFB site, died          between rat and human.
by apoptosis within three days, in the presence or absence of 1 nM         Whilst mouse models have utility in studying the effects of genetic
PDGF. Previous data demonstrated that cell death was correlated          manipulation on the healing and scarring response, and pigs have
with a metabolic block that was not abrogated by PDGF. More re-          clear utility in wound healing studies, this analysis demonstrates that
cently, we have discovered that FN-GFB sites must be present on          for scar reduction studies the rat represents the most suitable model
the same surface as FN-CBD to overcome this metabolic block.             in which to evaluate potential scar reduction therapies for man.
When FN-null cells were cultured on wells precoated with FN-CBD
and then stimulated with PDGF in the presence of either fluid phase      99
FN or recombinant FN-GFB domains cells died, whereas FN-null             degradation oF human collagenS By cloStridium
cells cultured on wells precoated with FN-CBD and either contigu-        collagenaSe and their eFFect on cell migration
ous or noncontiguous FN-GFB domains survived. Interestingly,             Lei Shi, PhD1, Ryan Ermis, BS1, Tasha Garcia1, Dale Telgenhoff,
when FN-null cells were cultured on type 1 collagen-coated wells         PhD2, Duncan Aust, PhD1.
and then stimulated with PDGF, they only survived when beads             1
                                                                          Healthpoint Ltd., Fort Worth, TX, USA, 2Tarleton State University,
coated with both FN-CBD and FN-GFB domains were added to the             Fort Worth, TX, USA.
culture, but not when beads were coated with only FN-CBD or FN-            Clostridium collagenase has been widely used in biomedical re-
GFB domains, even if the two sets of beads were added simultane-         search to dissociate tissues and isolate cells. Since 1965, the enzyme
ously. Thus fibroblast survival appears to require FN-GFB domain         has been used as a therapeutic drug* for the removal of wound

W38 2009 SympoSium on advanced Wound care and Wound healing Society meeting
necrotic tissues. Previous studies found that purified C. collagenase        Conclusions: These results suggest that PRFE treatment increases
could stimulate cellular response to injury and induce increases in        the expression of genes normally involved in fibroblast and ker-
cell proliferation and migration.3 The purpose of this in vitro study      atinocytes cell growth, tissue regeneration, and wound healing, po-
was to investigate the ability of C. collagenase to digest human col-      tentially contributing to the acceleration in wound healing observed
lagen Types I, II, III, IV, V, and VI (Types IV, V, and VI have not been   in vivo.
previously examined) and to determine the effect of C. collagenase
and collagen degradation products on the migration of keratinocytes        101
and fibroblasts. The gold surface migration assay was used for             continuouS Streaming oF therapeutic SolutionS
human keratinocytes, while a QCM method was used for human fi-             For the management oF chronic Foot ulcer
broblasts. Our results confirmed the digestion of human collagen           Amihay Freeman, PhD, Professor1, Tali Yaakobi, PhD1, Noa Hadar,
Type I, II and III by C. collagenase, and showed that C. collagenase       PhD1, Amir Shiner, BSc2, Sam Nitezki, MD3, Jonathan Lorber,
could also break down human collagen Type IV and V but not VI.             MD4, Arie Bass, MD, Professor4.
Among the collagens that could be degraded, the degradation prod-          1
                                                                            Tel Aviv University, Tel Aviv, Israel, 2EnzySurge Ltd, Rosh Haayin,
ucts of Type I and III demonstrated promotion of keratinocyte migra-       Israel, 3Rambam Hospital, Haifa, Israel, 4Asaf Harofeh Hospital,
tion, while the other collagens showed no effect when compared             Zrifin, Israel.
with untreated controls. Similar effects were seen in the fibroblast         purpose: The growing numbers of chronic ulcers present a huge
migration test, with the exception that type IV collagen degradation       challenge to currently employed labor intensive treatments. There
products also induced migration. C. collagenase itself was con-            is an urgent need in a new approach providing effective, user-friendly
firmed to have the ability to enhance cell migration in both assays.       treatment modality especially at the home-care settings. Here we
The data suggest that C. collagenase is effective in digesting colla-      demonstrate feasibility, safety and preliminary efficacy of a new
gen debris in necrotic tissues, and that both C. collagenase and the       modality of chronic wound management: Continuous Streaming of
degradation products of collagenolysis have the potential to pro-          Therapeutic solutions (CST).
mote cell migration. Therefore, collagenase treatment may provide            methods: CST was affected by continuous streaming of solutions
at least two potential mechanisms to enhance cell migration.               from a feeding reservoir onto ulcers occluded within DermaStream
* Santyl® Collagenase Ointment (Healthpoint Ltd.)                          (an FDA approved product). Streaming was affected by gravity and
1. Riley KN and Herman IM, J Burns Wounds, 2005;4:112-124                  used solution collected in a collecting-bag. This system also enabled
                                                                           mild negative pressure on the wound. A multi-centered, double
100                                                                        blinded, Phase I/II clinical trial was launched in two hospitals to test
the eFFectS oF a pulSed radio FreQuency electro-                           safety and preliminary efficacy of increasing concentrations of pro-
magnetic Field on gene eXpreSSion uSing co-cul-                            teolytic enzyme for venous ulcers debridement. Patients were
tureS oF human diploid FiBroBlaStS and                                     treated by 5-consecutive daily 6hrs-streaming sessions, followed by
KeratinocyteS in vitro                                                     standard changes of wet dressings for 3months follow-up. Stream-
John Moffett, Ph.D..                                                       ing of same solution devoid of enzyme served as control.
Regenesis Biomedical, Inc, Scottsdale, AZ, USA.                              results: 48 patients were enrolled, 36 treated with enzyme contain-
  introduction: We previously described the use of an electro-             ing solutions and 12 with solutions devoid of enzyme. ~75% -with
magnetic field (PRFE) treatment for accelerating wound healing             dose dependence- of the patients treated with enzyme (total expo-
in vivo. To determine the biological mechanisms associated with            sure time 30 hours) experienced debridement with exposure of gran-
PRFE treatment we have used an in vitro model consisting of                ulation tissue. ~75% -with dose dependence- of debrided wounds
human dermal fibroblasts (HDF) and epidermal keratinocytes                 exhibited spontaneous wound-closure or substantial wound size re-
(HEK). We addressed the pattern of gene expression of HDF and              duction within 2-7 weeks post treatment. Patients with pains experi-
HEK cells in co-culture after treatment with PRFE using cDNA               enced pain relief from first session. Technical feasibility and user
micro array technology.                                                    friendliness of CST were demonstrated.
  materials and methods: HDF and HEK cells were cultured in mod-             conclusions: Our accumulating results indicate that CST is a safe
ified (serum-free media) as described by the supplier. Gene expres-        and effective modality for wound management, paving the way to
sion was determined using microarray analysis and RT-PCR. 1,176            its use as effective, user-friendly treatment at the outpatient clinic
cDNA sequences coding for growth factors, cytokines, transcription         and homecare settings, aiming at shorter healing time.
factors and proteins involved in growth, adhesion, and DNA replica-
tion were assayed for expression after PRFE treatment.                     102
  results: We found significant differences in gene expression in          Short-term integration oF human Bilayered living
co-cultures when compared to studies performed with single cell            SKin SuBStitute (apligraF®) graFted onto nude mice
type tissue cultures alone. These co-culture experiments suggest           Cecile F. Rousseau, PhD1, Fabienne Thillou2, Aurelie Pagnon-
that changes in gene expression are better models to study the             Minot3, Sylviane Guerret, PhD2, Daniel J. Hartmann, PhD3, Vin-
process of wound healing and regeneration in tissue. Our results           cent Ronfard, PhD4
suggest using co-cultures of HDF and HEK will produce an in                1
                                                                            Organogenesis Inc., Canton, MA, USA, 2Novotec, Lyon, France,
vitro model to determine the effects of PRFE treatment in a tem-           3
                                                                            University Claude Bernard Lyon 1, Faculty of Pharmacy, Labora-
poral and spatial manner. The co-culture in vitro model indicates          tory, Lyon, France, 4Organogenesis, Canton, MA, USA.
that after PFRE treatment of cells there is cellular communication           Apligraf® is a bilayered living skin substitute composed of a bovine
between the HDF and HEK which regulates gene expression.                   collagen lattice containing living human neonatal cells. Fibroblasts

                    2009 SympoSium on advanced Wound care and Wound healing Society meeting W39
underlay a fully differentiated epithelium composed of human                cantly accelerated the re-epithelialisation of wounds at the 600mM
keratinocytes. The analysis of Apligraf has shown that it contains          dose. The human study demonstrated a statistically significant im-
not only bovine type I collagen but also human types I, III and IV          provement (p<0.05) in healing with the 300mM dose, with a trend
collagens before and after implantation. To investigate remodel-            towards accelerated healing also observed at the 400mM dose. In-
ing over time, Apligraf was applied to a full thickness skin wound          tradermal administrations of Mannose-6-Phosphate were safe and
on athymic mice.                                                            well tolerated.
  Wound healing was followed using digital photography and image              These data demonstrate that intradermal administration of Man-
analysis software. After 2 and 6 weeks, biopsies were analyzed              nose-6-Phosphate accelerates acute wound healing, and high-
using histological staining, immunohistochemistry with human-spe-           light Mannose-6-Phosphate as a potential therapeutic for this
cific antibodies, and electron microscopy techniques, to examine the        clinical indication.
cellular and extracellular matrix components.
  Gross observations of the wound revealed no obvious host                  104
reaction. The area of the wound decreased 66% during the                    can a drug-Free dreSSing decreaSe inFlammation
first week, and to 80% at 6 weeks. Apligraf was well integrated             and Wound pain? What doeS the evidence Say?
and vascularized at 2 and 6 weeks. Host cells invaded the bi-               Roger Sessions, D.O..
layered living skin substitute from the edges. These experi-                Ferris Mfg. Corp., Burr Ridge, IL, USA.
ments also showed that human marker proteins could be                         Background: Inflammation often plays a critical role in wound for-
found for at least 6 weeks post implantation, but no inflamma-              mation, and it can inhibit healing. Wound pain not only diminishes
tory response was elicited. At 2 weeks, Ki67 labeling identified            patient quality of life, it also directly inhibits healing. Debilitated
active cell proliferation in the epidermis of the implant, espe-            wound patients are often poor candidates for systemic pharmaceu-
cially within the basal layer.                                              tical pain relievers. Topical pharmaceutical pain relievers and anti-in-
  We also observed an evolution of the dermal-epidermal junction            flammatory drugs increase infection risks and/or directly impair
within the implanted bilayered living skin substitute: initially the        wound healing.
basement membrane was discontinuous. By 2 weeks post im-                      This study examines the evidence behind claims that polymeric
plantation, it became continuous and well defined, with numer-              membrane dressings* can inhibit the nociceptor response, even
ous hemi-desmosomes.                                                        through intact skin, often providing a dramatic drug-free decrease
  The combination of different techniques of histology to assess tis-       in inflammation and wound pain. If these claims are valid, polymeric
sue morphology and ultrastructure lead to a better understanding            membrane dressings can diminish the negative effects of pain and
of the components and processes involved in the successful incor-           inflammation without the adverse consequences associated with
poration of Apligraf, which can help better understand the potential        pharmaceuticals.
impact of skin substitutes on acute wounds.                                   methodology: All peer-reviewed published data about polymeric
                                                                            membrane dressings were evaluated to determine the robustness
103                                                                         of the evidence concerning its nociceptor response inhibition.
mannoSe-6-phoSphate intradermal inJectionS ac-                                This included:
celerate Wound healing in preclinical and clinical                            • Four scientific studies: a controlled trial of pain and inflam-
modelS                                                                           mation following arthroscopy, a study including both human
Nick Occleston, BSc (Hons), MPhil, PhD, Neil French,                             burn patients and an animal model and two strictly animal
BSc(Hons), MSc, PhD, Abdul Sattar, BSc (Hons), MSc, PhD,                         model studies
Adam Boanas, BSc, Tracey Mason, BSc (Hons), PhD, David Fair-                  • Three major facility-based product evaluations in which pain
lamb, BSc (Hons), Mark Cooper, BSc (Hons), MSc, Jim Bush,                        and/or inflammation were discussed
MBChB, MRCS (Ed), Sharon O'Kane, BSc (Hons), PhD, Mark                        • 80 individual patient outcomes in published peer-reviewed
W. J. Ferguson, BSc (Hons), BDS, PhD, DMedSci..                                  case studies or series in which wound pain and/or inflamma-
Renovo, Manchester, United Kingdom.                                              tion were problematic and polymeric membrane dressings
  Pre-clinical studies evaluating the effects of intradermal adminis-            were used
tration of an aqueous formulation of Mannose-6-Phosphate on the               results: Laboratory research unequivocally concludes that
acceleration of healing were conducted in split-thickness skin graft        polymeric membrane dressings inhibit the nociceptor response
donor sites in male Large White Yorkshire pigs. The studies evalu-          at the application site and that this can result in significantly di-
ated the effects of varying doses of Mannose-6-Phosphate (200 to            minished pain and inflammation. The facility-based evaluations
600mM), with histological measures of wound re-epithelialisation as         found that pain, spasticity and bruising are decreased and patient
the study endpoint. Translation of these data to man were studied in        mobility is increased when polymeric membrane dressings are
a double-blind, within-subject, placebo controlled clinical trial in male   used. 92.5% of the patients in the peer-reviewed case studies
and female volunteers aged 18-45 (a total of 70 subjects). This in-         using polymeric membrane dressings experienced a reduction
volved punch biopsies and investigated acute intradermal injections         in inflammation and/or wound pain.
(once at the time of wounding and once 24 hours later) of Mannose-            conclusion: Polymeric membrane dressings definitely can in-
6-Phosphate at doses of 50 to 600mM, with histological measures             hibit the nociceptor response at the application site. This has ex-
of wound re-epithelialisation as the primary endpoint.                      citing implications for both pain relief and wound healing.
  Preclinical studies in pigs demonstrated that acute administrations       *PolyMem
(once at the time of wounding) of Mannose-6-Phosphate signifi-

W40 2009 SympoSium on advanced Wound care and Wound healing Society meeting
105                                                                                            abstracts 107 - 189
evidence For Wound therapieS - in vitro, in vivo, in
                                                                                             poster general Session
Derek Silcock, PhD.                                                                         monday april 27, 7:30 - 9:00
Systagenix Wound Management, North Yorkshire, United King-
dom.                                                                        107
  There is much interest and debate around the need for clinical evi-       eFFect oF dreSSing changing on Wound healing
dence to support the selection and utilisation of wound care prod-          Study in a raBBit ear punch model
ucts. However, there is also much debate around what qualifies as           Yanan Zhao, M.D., Shengxian, Thomas A. Mustoe
sufficient evidence; evidence-based medicine has its origins in the         Northwestern University, Chicago, IL, USA.
conscientious and judicious use of medical research, combined with            The rabbit ear punch model is heavily used in wound healing and
clinical expertise and patient values, applied to the management and        hypertrophic scarring studies. As an important part of the protocol,
treatment of patients. There are many types of evidence, from in vitro      a semi-occlusive polyurethane film (Tegaderm) is placed on the
laboratory data to Randomised Clinical Trials (RCT) that are avail-         wounds and kept in place until the time of harvest or the wounds
able to support the use of products for medical conditions. While           have re-epithelialized, with reapplication as needed (i.e. if the Tega-
RCTs are deemed the ultimate statistical proof that a product is sig-       derm has fallen off the wound but the wound is still without infection
nificantly better than another treatment, they are rarely undertaken        or dessication). The goal of this study is to test the effect of dressing
with chronic wound therapies due to patient heterogeneity and cost.         changing on wound healing with a rabbit ear punch model. Two
As a result advanced therapies are often poorly adopted due to the          young New Zealand White rabbits were used in this study. Four 7-
lack of available evidence to support their cost.                           mm full-thickness dermal punches were then made on the inner sur-
  If however, we consider the hierarchy of clinical evidence it is clear    face of the ear down to perichondrium. One ear in each rabbit served
that multiple levels of evidence exist and that in combination they         as the control ear, which was covered by Tegaderm until the harvest
can increase our confidence in selecting which product to use. Evi-         day, while each wound in the other ear had Tegaderm changed at 3
dence needs to be clinically relevant though not necessarily clinically     and 6 days after wounding. At 7 days post wounding, the animals
sourced, and it should address the clinical need, not the competitive       were sacrificed and the wounds were collected and processed for
situation. Ideally, support data should be available from multiple lev-     histological analysis. Results: the dressing changing group had sig-
els of scientific and clinical investigation. Such data should, when-       nificantly less keratinocyte proliferation in terms of epithelial height
ever possible be published in peer-reviewed journals so that it has         and epidermal area than no dressing changing group, while the
been independently reviewed and deemed to be scientifically valid.          dressing changing group had less reepithelialization in terms of ep-
  In this study we have reviewed the different types of evidence that       ithelial gap and percentage healed and less granulation tissue for-
have been published for collagen-based wound care products,                 mation in terms of granulation tissue distance with marginal
specifically those intended for the treatment of chronic wounds.            evidence. Conclusion: the replacement of the semi-occlusive dress-
                                                                            ing is not a minor issue, which can give false positive or false nega-
106                                                                         tive results if not monitoring closely when carrying out wound
a murine model For BioFilm inFection in partial                             healing study.
thicKneSS WoundS
Paul Renick, MS, Eric Roche, PhD.                                           108
Healthpoint Ltd., Fort Worth, TX, USA.                                      characteriZing tiSSue engineered cellS over time
  A mouse model of infection in partial thickness wounds was devel-         Judith Fulton, PhD, Denise Blanco, BS-MT, Mark Tornero, BS.
oped in which the infection has biofilm characteristics. A partial thick-   Akron General Medical Center, Akron, OH, USA.
ness wound was generated on the back of each mouse and wounds                 The long-term goal of this study involves the creation of skin equiv-
were inoculated with a multi-drug resistant MRSA strain and cov-            alents with improved regenerative capacities by maximizing the re-
ered with a bandage. The resulting infection exhibited high bacterial       tention of resident stem and proliferating cells. A fresh harvest of
levels in the tissue that were stable over the course of several days.      skin cells contains a variety of types which include hematopoietic
Qualitative comparison between infected and uninfected animals              cells, fibrocytes, macrophages, endothelial cells, adipocytes and
suggested that the wound infection led to increased inflammation,           neural cells, as well as fibroblasts and keratinocytes. To further com-
more crust formation, and delayed healing. When treatment of                plicate the mixture, cells within each lineage exist in different stages
wounds with topical antimicrobials was initiated 4 hours after inoc-        of differentiation and release signals that influence the other cells.
ulation, several topical agents exhibited a significant reduction in the    Such interdependent activity enhances the chance that isolating sin-
bacterial load of the wound tissue. In contrast, when the infection         gle cell types will alter their phenotypes and result in unwanted en-
was allowed 24 hours to establish, the effect of topical agents was         vironmental selections.
greatly reduced. Microscopic examination of stained histology sam-            Neonatal foreskin keratinocytes and fibroblasts were collected
ples revealed the presence of a dense staphylococcal community.             from fresh tissue and used cultured or uncultured. The cells were se-
The observed bacterial community and the lack of drug efficacy              quentially seeded onto a cross-linked collagen matrix, and after in-
strongly support the biofilm nature of this model. The model should         cubation at the air-liquid interface, the resultant bilayers were
serve as a valuable tool for studying biofilm infection and identifying     processed for hematoxylin and eosin staining and immunohisto-
novel agents for prevention or treatment of wound biofilms.                 chemistry. Also analyzed, were fresh cell suspensions cytospun onto
                                                                            slides and cells cultured for various time periods.

                    2009 SympoSium on advanced Wound care and Wound healing Society meeting W41
  Use of fresh epidermal cells as well as fresh dermal cells resulted        create long time survived rabbits for wound study.
in a thicker, more stratified and differentiated epidermis. In normal          methods: 1. Alloxan (150 mg/kg) was given intravenously to 10
skin, cytokeratin 10 (CK10, marker for differentiating keratinocytes)        young New Zealand rabbits. Glucose was provided for 2-3 days. An-
stained cells in the suprabasilar layer of the epidermis, while integrin-    other 12 normal rabbits were used for comparison.
α1 (stem and proliferating cells) stained the basal layer. Fresh suspen-       2. Insulin (1-4 units/kg) was given daily after diabetes mellitus was
sions of epidermal cells contained both CK10 and α1 staining cells,          developed.
while cultured cells stained only for α1. In addition, the epidermal layer     3. Blood glucose was measured daily then weekly. Blood
of freshly engineered skin bilayers stained for α1 but not CK10.             chemistries were measured weekly and monthly.
  In conclusion, the composition of cells harvested from fresh skin            4. An insulin-glucose profile was made for each rabbit to balance
changes over time in culture, and this in turn, affects the quality of       hyperglycemia and overall health.
engineered skin equivalents. The next step will involve following the          5. Wounds were made on the ears at different times and healing
fate of cells over time in culture on a variety of matrices with the goal    was compared.
of retaining a variety of cells.                                               6. Six rabbits were sacrificed at 9 or 12 months and histology and
                                                                             molecular biological studies were performed.
109                                                                            results: 1. Hyperglycemia (<500 mg/dl) developed within 48 hours
the relationShip oF chronic venouS diSorderS to                              after alloxan.
inJection drug uSe                                                             2. All the animals developed the typical signs of diabetes gradually.
Barbara A. Pieper, PhD, RN1, Thomas N. Templin, PhD1, Robert S.                3. All the diabetic animals gained some weight but the gain was
Kirsner, MD, PhD2, Thomas J. Birk, PhD, MPT1.                                much less than the age-matched non-diabetic rabbits.
 Wayne State University, Detroit, MI, USA, 2University of Miami,               4. Hypertriglyceridemia, increased blood urea nitrogen and creati-
Miami, FL, USA.                                                              nine, and ketoacidosis were found in some of the diabetic animals.
  purpose: This study examined for injection drug users the distribu-          5. Histologically, the dorsal and ear skin showed the neuropathic
tion and severity of chronic venous disorders (CVD) and the impact           and microvascular changes. The pancreas showed marked beta cell
of years of injecting in the lower extremities on severity of CVD.           damages and thickened arterial walls. The kidney showed signifi-
  methods: The study was a cross-sectional, comparative design               cantly thickened afferent glomerular arterioles and narrowed lu-
stratified by age, sex, ethnicity, and 3 types of drug use (non-injection;   mens along with glomerular atrophy. There were lipid accumulations
arm or upper body injection only; and legs with or without upper             in the cytoplasm of the hepatocytes as vacuoles.
body injection). Participants (N=713) completed demographic,                   6. There was no difference in wound healing times between 2-week
health and substances abuse questionnaires and were evaluated                diabetic rabbits and non-diabetic rabbits. Wound healing was de-
using the clinical component of the Clinical-Etiology-Anatomy-               layed gradually in the rabbits with 3-month or longer diabetic times.
Pathophysiology (CEAP) Classification.                                         conclusion: With careful management, alloxan-induced diabetic
  results: Persons who injected in the groin, legs, and feet had sig-        rabbits can be kept for a year with reasonably good health for wound
nificantly worse CVD than those who never injected and those who             study. Wound healing was delayed only in longer diabetic animals.
injected in the arms/upper body. Thirty-nine percent of leg injectors
versus 4.2% or non-injectors or arm only injectors had CVD in the            111
moderate to severe categories. After controlling for age and co-mor-         Four patientS With chronic leg ulcerS under treat-
bid conditions, persons who injected in the legs were 9.14 times             ment With hydroXycarBamid - treatment Strategy
more likely to develop venous ulcers than those that injected in the         and outcomeS
arms and upper body only and 34.64 times more likely as those who            Claas Luedemann, Berthold Amann.
never injected. Analysis of the clinical CEAP identified one symptom         Franziskus Krankenhaus, Berlin, Germany.
cluster consisting of vein changes not associated with advanced dis-           Chronic leg ulcers induced by hydroxycarbamid, a cytostatic drug
ease (telangiectasis, reticular veins, malleolar flare and varicose          used in the treatment of certain myeloproliferative diseases, are dif-
veins) and another consisting of pigmentation, eczema, and lipoder-          ficult to treat. Although skin complications like erythema are quite
matosclerosis, and active or healed ulcers. Edema was equally as-            common, chronic ulcers are rarely mentioned in the literature. From
sociated with both symptom clusters. Conclusions: CVD is                     what we know it seems to be important to stop the hydroxycarbamid
associated with injecting in the groin, legs and feet as compared to         medication and switch to another drug (for example anagrelid). We
other sites. Pigmentation, eczema, and lipodermatosclerosis, and             report of 4 patients with such ulcers we treated in our hospital.
active or healed ulcers were represented the symptom cluster of ad-            2 patients were female (age 80 and 84 years, 1 with essential
vanced disease. Funding: National Institute of Nursing                       thrombocytosis (ET), one with polycythaemia vera (PCV)), 2 male
Research/National Institute of Health (NINR/NIH), R01 NR009264.              (72 and 81 years, one ET, one PCV), they were on hydroxycarbamid
                                                                             for 1 to 2.5 years. All of them had an ulcer of the ankle (since 1 to 14
110                                                                          months) without any other cause but the hydroxycarbamid medica-
long-term diaBetic raBBitS For Wound reSearch                                tion detectable. A heavy pain due to the wound was present in all pa-
Jianpu Wang, MD PhD, Rong Wan, MD PhD, Yiqun Mo, MD                          tients and made a complex pain medication necessary. Every wound
PhD, Ming Li, BS, Qunwei Zhang, MD PhD, Sufan Chien, MD.                     had signs of infection so that an antibiotic treatment was started. Hy-
University of Louisville, Louisville, KY, USA.                               droxycarbamid was discontinued and the patients received anagre-
  purpose: Short lived diabetic rabbits (<2 months) have severely            lid. Sharp debridement was performed several times in each patient,
limited the diabetic wound research capabilities. This study was to          2 were treated with maggot therapy, 3 with topic negative pressure

W42 2009 SympoSium on advanced Wound care and Wound healing Society meeting
therapy to induce granulation. 3 of the 4 patients underwent a split-       introduction: Cervical fistulae caused by unsuccessful wound heal-
skin transplantation (1 patient rejected this option). After 4-5 month    ing after microsurgical reconstruction are sometimes seen in patients
all 3 wounds after skin transplantation were closed, the one without      who have undergone radical ablation of head and neck malignancies.
transplantation was significantly reduced in size. Only 1 patient was     These undesirable complications bring continuous distress for the
in need for pain medication sometimes.Altogether ulcers induced           patient and decrease their quality of life. Furthermore, treatment of
by hydroxycarbamid seem not to be as rare as described in literature.     fistulae is challengeable because these patients often treated with
The treatment of these wounds is a complex and long lasting               radiotherapy.
process and the cessation of hydroxycarbamid is necessary for heal-         methods: Eighty-three patients with head and neck cancer who re-
ing. By skin transplantation wound healing can be accelerated. After      quired radical resection and microsurgical reconstruction in our unit
wound closure most of the patients seem to be free of pain.               from 2004 through 2007 were eligible for this study retrospectively.
                                                                          Of these patients, 11 developed cervical fistulae postoperatively. We
112                                                                       investigated influence of radiation, chemotherapy, size of tumors,
a retroSpective analySiS oF a collagen matriX                             lymph node metastasis, and type of flaps on development the post-
dreSSing With Silver* compared to an eXtracellu-                          operative fistulae.
lar Wound matriX dreSSing in the treatment oFve-                            results: All of 11 patients who developed cervical fistulae were
nouS leg ulcerationS                                                      treated with radiography, which was thought to be the most affective
Karre Hermann, RN, MSN, APNP, Jeffrey Niezgoda, MD, FACHM,                factor on occurring the postoperative cervical fistulae. All the fistulae
FACEP.                                                                    were successfully and reliably treated with the pectoralis major mus-
Hyperbaric and Wound Care Associates, Milwaukee, WI, USA.                 clocutaneous flap.
  Chronic venous ulcerations are costly from a socio-economic               conclusions: Cervical fistulae after radiation with microsurgical re-
standpoint. It is estimated that 1% of the world’s population will ex-    construction will not heal spontaneously despite aggressive medical
perience venous ulcerations and incur treatment costs of more than        wound management. Skin grafts and local cutaneous flaps located
$3 bilion(1). Treatment of chronic nonhealing wounds is aimed at re-      within the radiation field are unreliable and rarely provide adequate
ducing venous reflux and supporting normal endogenous wound               stable coverage. Salvage surgery with musclocutaneous flap is an
healing processes. Chronic wounds are those that have been pres-          only recommended method to heal these complex wound.
ent for more than 6 months and fail to progress along the normal
stages of wound healing. Wounds that fail to progress from the pro-       114
liferative stage may be due to a lack of extracellular matrix. Adding     epidemiology and economicS oF a vulneraBle pop-
an extracellular matrix has been shown to increase granulation tis-       ulation Four yearS later
sue and facilitate epidermal migration(2).                                Gayle Harrell, Barbara P. Rogers, Annette B. Wysocki.
  A retrospective analysis was carried out on 15 patients with chronic    University of Mississippi Medical Center, Jackson, MS, USA.
venous leg ulcerations. All patients were treated at the Center for         Vulnerable populations often experience decreased access to
Comprehensive Wound Care and Hyperbaric Medicine, Milwaukee,              health care and are an integral feature of many academic medical
WI between 2004-2008. Patients were treated with either a collagen        centers. We previously reported on the epidemiology and economics
matrix dressing with silver* or with an extracellular wound matrix**      of a vulnerable population after one year of the opening of a new
dressing for at least four weeks, or until ulcer closure. Compression     wound clinic directed and operated by a nurse practitioner in a mi-
bandages were standard for the institution with either a 3-4 layer        nority serving institution. Here we report on the profile of this popu-
compression bandage***. Patient demographics, medical history,            lation after four years of operation during which time the practice and
and wound details were all recorded in an electronic database. The        referral patterns have become established. Currently 34% of our pop-
size of the wound, tissue type, level of exudate, odor, and pain levels   ulation is over 60 years of age; 46% are between 41-59 years of age;
were also recorded. Efficacy was determined by comparing the rate         and 20% are 40 years of age or under compared with 48%, 45% and
of wound healing and/or the percentage reduction in wound area            7% respectively at one year. The vulnerable population in the clinic is
over time.                                                                now composed of 64% African Americans; 2% Native Americans;
  The full results of our analysis will be presented in a poster dis-     1.3% Hispanic; and 32% Caucasian compared with 51.6%, 6.5%, 3.2%
cussing the most cost effective and efficacious of the two dressings.     and 38.7% at one year. The etiologies of these wounds is now 50.5%
*Biostep AG - Smith & Nephew Wound Management Inc., St. Petersburg,       diabetic/neuropathic and 26.8% venous compared with 31% and
   FL                                                                     28.5% previously with the remaining 22.7% being oncologic, ortho-
**OASIS Wound Matrix - Healthpoint Ltd, Fort Worth, TX.                   pedic surgery, decubitus and other. In general, the total number of
***PROFORE Multi-layer bandage system - Smith & Nephew Wound              clinic visits has risen with 39% having 2 to 5 visits; 31% 6 to 25 visits;
   Management Inc., St Petersburg, FL
                                                                          5% 26 to 49 visits; and 3% having 50 or more visits. Our current payer
                                                                          mix is 56% either Medicare or Medicaid, or a combination, while the
                                                                          remainder are covered by private insurance (20%); workman’s com-
evaluation and treatment oF cervical FiStulae
                                                                          pensation (1%); or self-pay (23%) compared with 61%, 23%, 3.2% and
aFter microSurgical reconStruction FolloWing
                                                                          12.9% previously. Thus, our data indicate that the population we serve
radical aBlation oF head and necK cancerS
                                                                          is somewhat stable over time with a noticeable increase in the per-
Masaki Fujioka, MD,PhD, Kiyoshi Oka, MD, Riko Kitamura, MD,
                                                                          cent of diabetic/neuropathic wounds (31% v. 50.5%), in the number
Aya Yakabe, MD.
                                                                          of visits over six required to manage or achieve wound healing (30%
National Hospital Organization Nagasaki Medical Center.,
                                                                          v. 39%), and in the number of self-pay individuals (12.9% v. 23%).
Ohmura, Japan.

                    2009 SympoSium on advanced Wound care and Wound healing Society meeting W43
115                                                                        during wound healing. Parabiotic pairs have been surgically created
three-dimenSional tiSSue modelS oF FiBroplaSia: eX-                        consisting of combinations of collagen promoter (marker of dermal
tracellular matriX-dependent proteaSe and pro-                             fibroblasts) GFP transgenic mice and smooth muscle actin (marker
teaSe inhiBitor eXpreSSion By human SKin                                   of myofibroblasts) GFP transgenic mice. During parabiosis surgery,
FiBroBlaStS                                                                the incision was made on the flank of each mouse and the pair was
Tai-Lan Tuan, PhD1, Paul Benya, PhD2, Yang Ping Han, PhD3, An-             then sutured together to establish a common blood circulation be-
nette Wysocki, PhD4.                                                       tween parabionts. At sacrifice frozen skin sections were prepared
 The Saban Research Institute of Childrens Hospital Los Angeles,           from the region where mice were surgically joined. Since mice con-
Department of Surgery, Keck School of Medicine, University of              tained transgenes with different GFP isoforms, we were able to track
Southern California, Los Angeles, CA, USA, 2Orthopaedic Hospi-             cells originating from either of parabionts. Histological preparations
tal, Los Angeles, J. Vernon Luck, Sr., M.D. Research Center and            of the connection area between parabiotic animals revealed the
UCLA-Orthopaedic Hospital Department of Orthopaedic Surgery,               sharp border of GFP expression, thus separating dermal fibroblasts
David Geffen School of Medicine at UCLA, University of Califor-            and myofibroblasts that originated from different animals. There was
nia, Los Angeles, California, USA, Los Angeles, CA, USA, 3De-              very little mixing of fibroblasts and myofibroblasts originating from
partment of Surgery, Keck School of Medicine, University of                different animals. This data suggest that only fibroblasts and myofi-
Southern California, Los Angeles, CA, USA, 4University of Missis-          broblasts of local origin contribute to the wound healing process. In
sippi Medical Center, Jackson, MS, USA.                                    addition, the parabiotic model using transgenic mice that mark de-
  The development of 3-dimensional tissue models that integrate es-        fined cell populations is a novel approach to study local migration
sential cell types, instructive ECM scaffolds, and spatial and physical    and interaction of cells during wound healing.
elements is fundamental to the understanding the intricate process
of organ function and repair. The current study is carried out to in-      117
vestigate major proteases and their inhibitor expressions by fibrob-       chemoKineS and Wound healing in diaBetic Foot
lasts during fibroplasia using physiologically relevant 3-dimenional       WoundS: macrophageS, macrophage chemoattrac-
extracellular matrix (ECM) gels composed of fibrin, collagen, or mix-      tant protein 1 and vaScular endothelial groWth
tures of fibrin and collagen to simulate ECM composition at various        Factor under Standard treatment verSuS treat-
stages of the repair process. The results show while the level of col-     ment With a matriX metalloproteinaSeS inhiBitor
lagen synthesis by fibroblasts cultured in restrained fibrin or collagen   (promogran®)
gels is similar over a 10 day period, the expression of plasminogen        Claas Luedemann1, Shanti Naskar2, Berthold Amann1.
activator (PA) and matrix metalloproteinase (MMP) are different and        1
                                                                            Franziskus Krankenhaus, Berlin, Germany, 2Gefäßkrankheiten
the expression of active urokinase PA (uPA) is correlated with active      Essen, Essen, Germany.
MMP-2. Subsequent investigations using fibrin-collagen hybrid gels           Wound healing is a complex process in which macrophages play
and perturbation studies indicate that MMP-2 activation is mediated        a major role. We know that macrophage chemoattractant protein 1
not by uPA/plasmin but by another species of MMP, possibly MT1-            (MCP-1) is the major human macrophage attracting protein and vas-
MMP. The expression of plasminogen activator inhibitor-1 and tissue        cular endothelial growth factor (VEGF) is the main angiogenic factor
inhibitor of metalloproteinase-2 is both decreased as the content of       in the granulation phase of wound healing. Elevated matrix metallo-
collagen in the matrix increases. These findings demonstrate differ-       proteinases (MMPs) content in chronic wounds may impair secre-
ential regulation of the major ECM degrading proteases and their           tion and action of MCP-1 and /or VEGF thus impairing macrophage
inhibitors in 3-dimensional models of fibroplasia simulating injury re-    immigration and activity.
pair.                                                                        We immunohistochemical quantified macrophages (by CD68 de-
[Support: The National Institute of Health grant R01GM055081 (T.-L. T)]    tection), did immunoassays of MCP-1 (ELISA, monoclonal) and
                                                                           chemoluminescense assays of VEGF-1 at baseline and after 20 days
116                                                                        of treatment in 21 chronic diabetic foot wounds (11 with best stan-
aSSeSSment oF dermal FiBroBlaSt and myoFiBroB-                             dard care, 10 with a MMPs inhibitor (Promogran®)). The mean con-
laSt migration during Wound healing                                        centration of MCP-1 in untreated chronic diabetic foot wounds was
Tatjana Barisic-Dujmovic, MD, Stephen Clark, PhD.                          about 9.000 pg/ml, that of VEGF-1 about 3.000 pg/ml. In the granu-
University of Connecticut Health Center, Farmington, CT, USA.              lation phase of wound healing the MCP-1 concentration was low-
  Wound healing is a complex physiological response to the tissue          ered (compared to the findings in the chronic phase) to 35% in the
injury. In addition to the hematopoietic cells that participate in the     best standard care group compared to 31% in the MMPs inhibitor
early phases of the wound healing, dermal fibroblasts and myofi-           group (not significant). The VEGF-1 concentration rose to 126%
broblasts play a major role in the wound healing process. These cells      (standard care) compared to 185% (MMPs inhibitor, not significant).
synthesize the extracellular matrix and are responsible for wound          The macrophage density rose to 180% (standard care) compared to
contraction. It is unknown how these cells interact with each other        250% (MMPs inhibitor, p<0.05).
during wound healing. We have chosen to study the parabiosis                 Unlike expected, in chronic diabetic foot wounds an initial high con-
model, specifically the skin region where two mice are joined to each      centration of MCP-1 is present. The amount of MCP-1 shows a tight
other surgically. Since transgenic mice have been developed to mark        negative correlation with improvement in wound healing. The
populations of fibroblasts and myofibroblasts and they carry differ-       changes of MCP-1 and VEGF-1 levels showed no significant differ-
ent green fluorescent protein (GFP) colors, study was initiated to de-     ences between best standard care and the treatment with a MMPs
termine the local migration of dermal fibroblasts and myofibroblasts       inhibitor. The increase of macrophage density was influenced posi-

W44 2009 SympoSium on advanced Wound care and Wound healing Society meeting
We hypothesize that the acute inflammation, particularly the in-      wound dressings were comparable. The wounds responded well
flammatory cytokines, may coordinate this process, in part,           to each of the treatment applied and all new regenerated tissue
through epithelial-to-mesencymal transition. Furthermore, un-         appeared healthy with collagen and new vessel formation. The
resolved inflammation may maintain the elevated EMT into fi-          two NPWT dressing systems available on the market have been
brosis in scars. To these regards we first examined the               compared within the range of their clinically recommended pres-
association of inflammation with EMT in wound healing and fi-         sure levels which is -10kPa / -75mmHg for gauze and -20kPa / -
brosis. In developing stage of scars many basal epithelial cells      150mmHg for foam. They exhibited clinically comparable 10-day
were often observed in detached manner from basement mem-             healing rates. To conclude, there exist numerous clinically-effec-
brane. These epithelial cells exhibited mesencymal features as        tive alternatives, each having its own set of unique advantages
evidenced by elevated vimentin and FSP1 staining. Real time           (i.e. cost, ease-of-use, and pain management).
RT-PCR analysis also demonstrated the association of unre-
solved inflammation (TNF-alpha and many MMPs) to EMT fea-             183
tures (down-regulation of E-cadherin and up-regulation of             SKin tranSplantation aFter topic negative preS-
vimentin and FSP1) in scarring tissues. To recapitulate the EMT       Sure therapy improveS the outcome in chronic
in vitro we treated explants of normal human skin with inflam-        WoundS
matory cytokines or TGF-beta, a known fibrotic promoter. TNF-         Claas Luedemann, Münevver Güner, Berthold Amann.
alpha or TGF-beta unequivocally induced EMT as evidence by            Franziskus Krankenhaus, Berlin, Germany.
expression of vimentin and FSP1 in epithelial cells. Under the          There is little data available about long-term outcomes of pa-
cytokine treatment the primary human keratinocytes gained             tients with chronic wounds after topic negative pressure wound
mesencymal markers and lost E-cadherin, resulting in migration.       therapy (NPWT), especially in combination with skin transplan-
Moreover, the TNF-alpha-provoked EMT was association with             tation (ST). We are presenting the data of 71 patients with chronic
elevated MMPs; and conversely, MMP inhibitors impaired the            wounds (no response to best standard care) treated with NPWT,
EMT process. Thus, we demonstrate that the inflammatory cy-           46 of those combined with skin transplantation.
tokine may directly activate keratinocytes through EMT in               34 patients were female, 37 male, mean age 69 years, 39 with
wound healing and fibrosis.                                           type 2 diabetes mellitus. All with a chronic wound on the lower
                                                                      leg/foot with a mean wound size of 59 cm², 12 of them due to crit-
182                                                                   ical limb ischemia (CLI), 20 chronic venous insufficiency (CVI),
a compariSon oF variouS Wound dreSSingS cou-                          21 diabetic polyneuropathy (PNP) combined with CLI, 6 CLI and
pled to a negative preSSure Wound therapy                             CVI, 12 because of other reasons like vasculitis or superinfected
(npWt) SyStem to Study eFFectS on Wound healing                       trauma. All were treated with NPWT to induce granulation with a
progreSSion                                                           mean total NPWT duration of 13 days, 46 afterwards were skin
Rey Paglinawan, M.Sc.1, Miodrag Colic, M.D., Ph.D.2, Maryline         transplanted (33 with total skin pinch grafts, 13 with split skin
Simon, Ph.D.1.                                                        grafts) and immediately after ST received another cycle of NPWT
  Medela AG, Baar, Switzerland, 2Institute for Medical Research,      to optimize transplant healing. The 25 patients not treated with
Military Medical Academy, Belgrade, Serbia.                           ST received advanced moist wound therapy (Ant healing. The 25 pat
  aim: A growing number of evidence is accumulating showing                                            ST received advanced moist wound th
the beneficial effects of negative pressure wound therapy                                                                              ST receive
(NPWT) in wound healing. However, none have investigated the
optimal pressure level intensity, manner of delivery of negative
pressure together with various combinations of existing dressing
technologies. Various commercially available dressings (Gauze
type 1, Gauze type 2, Foam type 1, Foam type 2, Textile and Silver)
were tested in combination with a NPWT system applied with a
range of negative pressure levels. A number of wound healing
parameters were subsequently assessed.
  methodology: Landras type pigs were allowed to acclimatize to
the housing conditions prior to all procedures. The animals were
anesthetized, shaved and scrubbed in preparation for the oper-
ating room. Full thickness wounds extending down to the muscle
fascia (3.5 cm x 3.5 cm, maximal 10 per animal) were excised on
each pig. The wounds were filled with two types of gauze, two
types of foam, silver and textile, and coupled with NPWT for 10
days. This was done with negative pressures -10kPa (-75mmHg),
-20kPa (-150mmHg) and -30kPa (-225mmHg). Along with stan-
dard wound measurements taken previously, granulation tissue
area and other additional measurable wound healing effects of
NPWT are shown for the first time.
  result & conclusion: The 10-day healing rates from the various
  purpose: Diabetic wounds are deficient in the chemokine SDF-         Our results indicate that full length soluble CD109 and peptides
1α. Correction of this deficiency by the local application of mes-     corresponding to the putative TGF-beta1 binding region of
enchymal stem cells (MSCs) or the overexpression of SDF-1α can         CD109 inhibit TGF-beta1 binding to its receptors and TGF-beta
correct the wound healing defect in diabetic mice. The mecha-          signaling in vitro. Furthermore, surface plasmon resonance
nism by which this occurs has not been clearly elucidated and is       studies indicate strong cooperative binding characteristics be-
likely multifactorial. One potential mechanism of this correction      tween CD109 and TGF-beta1 in vitro. Together, our results
is the alteration of ECM proteolysis. We hypothesized that MSC         demonstrate that full length soluble CD109 and CD109 pep-
or lenti-SDF-1α treatment corrects the diabetic wound healing im-      tides bind TGF-beta1 with strong affinity, inhibit TGF-beta1 bind-
pairment, in part, by altering the proteolytic milieu of the ECM.      ing to its receptors and decrease TGF-beta1 signaling. These
  methods: Diabetic (Db-/Db-) or non-diabetic (Db+/Db-) mice           findings provide a basis for a novel therapeutic approach in
were wounded with an 8mm dermal punch device and treated               which a soluble CD109 peptide may be used to modulate TGF-
with either 1 x 106 MSCs or 1 x 109 p.f.u lenti-SDF-1α vector. At      beta action locally to manipulate the scarring process.
days 3 or 7, overlying eschar was removed and a tear strip was
placed on the wound for 30 minutes. Protein was eluted off the         186
strip over 2 hours in 4°C with a Tris/NaCl/Brij 35 buffer (pH =        the eFFectS oF decorin and Biglycan on the
7.6) and stored at -80°C. MMP-9 concentrations were measured           human vocal Fold FiBroBlaSt
using a standard, colorimetric ELISA. Protein concentration for        Priya Krishna, MD1, Xiaoqu Lu, BS2, Yong Ho Bae, MS2, Alan
each sample was quantified to normalize levels of MMP-9.               Wells, MD, DMSc2.
  results: Levels of MMP-9 were significantly higher (p=0.01) in       1
                                                                         University of Pittsburgh/UPMC Mercy, Pittsburgh, PA, USA,
diabetic wounds compared to non-diabetic mice. Following MSC           2
                                                                         University of Pittsburgh, Pittsburgh, PA, USA.
or lenti-SDF-1α treatment, diabetic wounds had a reduction in lev-       introduction: Vocal fold scarring presents a clinical challenge
els of MMP-9 levels.                                                   with no optimal solution, with the vocal fold fibroblast of the lam-
  conclusion: Treatment with MSCs or lenti-SDF-1α was associ-          ina propria as the key player in orchestrating both normal healing
ated with a decrease in the proteolytic activity in the wounds. This   and scarring. The proteoglycan decorin has shown promise in
finding demonstrates a potential mechanism, whereby improved           combating scarring in other organ systems. The hypothesis of
ECM synthesis rather than degradation following treatment, may         this study was that decorin and a similar proteoglycan, biglycan,
be responsible for the observed wound healing correction.              would prevent wound contraction in an in vitro model of scarring.
                                                                         methods: Human vocal fold fibroblasts were harvested from
185                                                                    vocal fold tissue from the warm autopsy program. Fibroblasts
identiFication oF SoluBle cd109 aS a potential                         (passages 3-6) were then used in a dose-response in vitro scratch
anti-Scarring agent                                                    assay using decorin and other growth factors (EGF, HGF,TGF-
Carter Li1, Hahn Soe-Lin1, Mark Hancook2, Anie Philip1.                beta). Single-cell tracking studies using both matrix and soluble
  Divison of Plastic Surgery, Department of Surgery, McGill            decorin and biglycan, immunofluorescence and pcr array for ex-
University Health Centre, Montreal, QC, Canada, 2Sheldon               tracellular matrix genes were also performed.
Biotechnology Centre, McGill University, Montreal, QC,                   results: in vitro scratch assay results showed a significant dif-
Canada.                                                                ference between control and decorin 40ug/ml treatment groups
  Hypertrophic scarring is a pervasive medical problem which           showed a significant difference in wound closure with less
often occurs as a result of burn, trauma, or surgical injury. Of       wound
                                                                       track- closure in the treatment group (p=0.002). Single cell-
the multitude of factors that have been examined in the context               ing
                                                                       net dis- demonstrated significant decrease in overall total and
of hypertrophic scarring, transforming growth factor-beta (TGF-        decorin  tance traveled by cells in a treatment group of soluble
beta) exhibits a broad spectrum of effects on the scarring             (p <.05 20ug/ml and biglycan 20ug/ml by single factor ANOVA
process. It is well documented that administration of anti-TGF-        down- for both). PCR Array (SA Biosciences)™ indicated gene
beta1 antibodies or a decrease in the TGF-beta1/beta3 isoform          500 regulation of type I collagen (13 fold), integrin alpha 6-8 (23-
ratio can reduce hypertrophic scarring. Our group has identi-                fold), forty fold-up regulation of MMP-2.
fied a novel GPI-anchored protein CD109, which binds TGF-                conclusion: Decorin and biglycan decrease overall cell motility
beta1 in an isoform-specific manner and is a component of the          and down regulate several genes important for cell adhesion.
TGF-beta receptor system. Previous data from our group sug-            These data suggest these SLRPs may have therapeutic benefit
gest that CD109 released from cell surface (soluble CD109)             in vivo for vocal fold scarring.
can bind TGF-beta1 with high affinity and inhibit TGF-beta1
binding to TGF-beta signaling receptors. To explore the poten-         189
tial of CD109 as a TGF-beta1 antagonist, we examined the effi-         amnion-derived cellular cytoKine Solution
cacy of full length soluble CD109 and CD109 peptides to bind           (accS) increaSeS Keratinocyte and FiBroBlaSt mi-
TGF-beta1 and inhibit TGF-beta1 signaling in vitro. CD109 pep-         gration in in vitro Boyden chamBer migration aSSay
tides corresponding to the putative TGF-beta1 binding domain           M. Georgina Uberti, MD, Yvonne N. Pierpont, MD, Francis Ko,
of CD109 were generated using GST fusion proteins or were              BS, Charlotte A. Smith, MS, Martin C. Robson, MD, Wyatt G.
obtained commercially. Ability of the full length protein and its      Payne, MD.
peptides to bind TGF-beta1 was analyzed by radioligand bind-           Institute for Tissue Regeneration, Repair, & Rehabilitation, Bay
ing, affinity labeling and surface plasmon resonance studies.          Pines VA Health Care System, Bay Pines, FL, USA.

                   2009 SympoSium on advanced Wound care and Wound healing Society meeting W63
  introduction: Amnion-derived multipotent progenitor (AMP) cells       activity of either keratinocytes or fibroblasts through the mem-
appear to be useful as adjuvants in wound healing. AMP cells se-        brane determined the influence of ACCS and was compared with
crete a unique combination of cytokines and growth factors, known       neutral buffered solution as control. Cell migration was evaluated
as Amnion-derived Cellular Cytokine Solution (ACCS), including          at 24 hours by counting the number of cells in five high-power
PDGF, VEGF, Angiogenin, TGF-B2, TIMP-1, and TIMP-2. In the skin,        fields. Data were expressed as the mean + SE. Statistical signifi-
a cytokine network between mesenchymal and epithelial cells             cance was determined by the paired Student t-test.
tightly controls keratinocyte and fibroblast migration, proliferation     results: Cell viability remained high throughout the experiment.
and differentiation. To evaluate the influence of ACCS on the mi-       In the presence of ACCS, both keratinocytes and fibroblasts
gratory behavior of keratinocytes and fibroblasts, a dual Boyden        demonstrated a statistically significant (P<0.05) increase in mi-
chamber co-culture cell-migration assay was utilized.                   gration as compared with controls in both collagen- and fi-
  materials and methods: Human neonatal foreskin fibroblasts            bronectin-coated membrane assays.
and human skin keratinocytes were utilized. Cell migration was            conclusion: Keratinocyte and fibroblast migration are in-
assayed quantitatively utilizing a Boyden chamber with 8µm              creased significantly when stimulated by ACCS in the in vitro
pore size polycarbonate membrane coated with either collagen            Boyden chamber. This implies that these cell types, important in
or fibronectin. Cells placed in the upper compartment and               normal wound healing, may be influenced to accelerate migration
ACCS placed in the lower chamber were incubated at 37 de-               in wounds, thus possibly accelerating wound repair. Further stud-
grees C in an atmosphere of 5% CO2. Chemotactic migration               ies in vivo will expand this theory.

W64 2009 SympoSium on advanced Wound care and Wound healing Society meeting

Shared By: