bluuebunny/crossref_metadata_2025 huggingface dataset:
027 Fibrin Sealant Combined with Fibroblasts and PDGF Enhance wound Healing in Excisional Wounds
Authors: Mogford J. E. , Tawil B. , Mustoe T. A.
Published: March 2007

A number of strategies have been explored for the treatment of cutaneous defects such as protein growth factor or gene therapy approaches. Alternative approaches include dermal or dermal‐epidermal skin substitutes or clot substitutes such as fibrin sealants (FS’s). Here we test the fibrinogen‐thrombin formulation of fibrin sealant combined with fibroblasts and PDGF in wound healing models. Four formulations varying in fibrinogen and thrombin concentration were applied to full‐thickness biopsy wounds in the rabbit ear cutaneous wound‐healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 × 105 cells/wound) embedded in the fibrinogen component. At post‐wounding day 7, there was no difference in the diluted vs. non‐diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/ml fibrinogen, 167 U/ml thrombin; n = 10) that was 167%(p < 0.05) of the non‐diluted FS containing cells (50 mg/ml fibrinogen, 250 U/ml thrombin; n = 10). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone (p < 0.05) for each diluted formulation although no differences in this parameter was observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF‐B (3 μg; n = 4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over 2‐fold (p < 0.01). Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in Fibrin Sealant that is adjusted to balance hemostasis with promotion of wound healing processes.
033 Regulation of TGF‐β Responses by a Novel Accessory TGF‐β Receptor in Human Keratinocytes
Authors: Marcoux A. , Finnson K. , Philip A.
Published: March 2007

The potential of TGF‐β isoforms to regulate wound healing and scarring is well documented in animal models. Since TGF‐β action is likely to be regulated at the level of its cell surface receptors, we analyzed TGF‐β receptor profiles and regulation of TGF‐β signaling in human keratinocytes. We identified a novel cell surface TGF‐β1 binding protein of 150 kDa (r150) on human keratinocytes that interacts with the TGF‐β signaling receptors. Further characterization of r150 demonstrated that it is a GPI‐anchored protein, that it can be released from the cell surface by an endogenous phospholipase C, and that the released form can bind to TGF‐β1. Recent cloning of r150 revealed it to be a novel protein of 1428 amino acids. Our objective was to determine the functional significance of r150 in regulating TGF‐β responses in keratinocytes.

Affinity labeling of keratinocytes overexpressing r150 cDNA and immunoprecipitation stud‐ies using anti‐r150 antibodies show that the cloned cDNA represents r150. Importantly, over expression of r150 results in inhibition of TGF‐β1‐induced Smad 2 and Smad 3 phosphorylation, gene transcriptional activity, and keratinocyte migration. In contrast, loss of r150 function using antisense morpholino oligos of r150 leads to enhanced Smad 2 and Smad 3 phosphorylation, gene transcriptional activity and proliferation of keratinocytes. In summary, our results demonstrate that r150 is a potent inhibitor of TGF‐β signaling in keratinocytes, and that it may have potential therapeutic value in modulating TGF‐β action in human diseases where TGF‐β plays a pathophysiological role. As such, r150 may be of use in reducing hypertrophic scarring, and an r150 antagonist may promote wound healing.
036 Overexpression of Smad7 in Keratinocytes Accelerates Cutaneous Wound Healing
Authors: Han G. , Li A. G. , Owens P. , Wang X‐J
Published: March 2007

Transforming growth factor β(TGFβ) has both positive and negative effects on cutaneous wound healing. Smad7 acts as a major downstream antagonist of TGFβ signaling in keratinocytes and its role in wound healing has not been defined. We have established a Smad7 transgenic mouse line using a keratin 5 (K5) promoter (K5.Smad7) which expresses Smad7 transgene at a mild level (∼2 fold of the endogenous Smad7 in the skin). These mice did not have overt skin defects as shown from our previous Smad7 transgenic mice expressing much higher levels of the Smad7 transgene (EMBO J 2002, 21:2580–90). K5.Smad7 mice from the above low expressor line and non‐transgenic littermates were subject to 6‐mm full‐thickness excisional wounding. K5.Smad7 mice exhibited early scab rejection, reduced inflammation, and accelerated re‐epithelialization as compared to non‐transgenic mice. To further determine the stage‐specific effects of Smad7 on wound healing, we generated a transgenic model in which Smad7 transgene expression can be induced in the epidermis and hair follicles (gene‐switch‐Smad7) by topically RU486 application. Smad7 induction from day 3 to day 7 after excisional wounding reduced inflammatory responses through suppressing expression of a variety of inflammatory cytokines/chemokines in gene‐switch‐Smad7 mice as compared to control mice. Meanwhile, overexpression of Smad7 exhibited accelerated re‐epithelialization, which correlated with increased expression of metalloproteinases and elevated Erk (extracellular signal‐regulated kinases) signaling in the leading epidermal edges in gene‐switch‐Smad7 wounds compared to control wounds. Smad7 induction from day 7 to day 20 after excisional wounding reduced dermal fibrotic response and angiogenesis in the dermis, resulting in a better tissue repair. We conclude that the effectsof Smad7 on wound healing are likely due to blocking the negative effects of TGFβ on cutaneous wound healing.
037 Fibroblast growth factor – binding protein cDNA and truncated variants are active in diabetic wound healing
Authors: Dasgupta Jayasri , Bittner Michaela , Goppelt Andreas , Werner Sabine , Beer Hans‐Dietmar , Davidson Jeffrey M.
Published: March 2007

Fibroblast growth factor – binding protein (FGF‐BP) is a secreted protein that appears to function as a low affinity heparin –binding protein. FGF‐BP binds to FGF‐1 and ‐2 in a non‐covalent, reversible manner to mobilize and solubilize these growth factors from their storage sites in the extracellular matrix. FGF‐BP is involved in both developmental and adult tissue homeostasis as well as in angiogenesis and tumorogenesis involving FGF‐1/2. FGF‐BP is overexpressed in several tumor types: head and neck, skin, cervical, and lung cancer, squamous cell carcinoma, and colon and breast adenocarcinoma. To establish the effect of FGF‐BP on wound healing, several forms of FGF‐BP cDNA were administered by particle‐mediated gene transfer into various animal wound models using the gene gun (Bio‐Rad). In a rat incisional wound model, gene gun cDNA delivery of full length FGF‐BP at the time of surgery produced a 117% increase of wound strength in diabetic rats at 10d, although the relative increase did not reach statistical significance (P < 0.08). Two truncated variants of FGF‐BP (pFGFbp10 and 17) were also administered in the rat incisional wound model by gene gun technique. pFGFbp17 increased the wound strength in diabetic rat 129%(p < 0.03), and the relative increase reach statistical significance (P < 0.008). In the rabbit ear ulcer model, particle‐mediated transduction of full length FGF‐BP increased collagen content by 195% and wound closure rate 38% at 10d post‐surgery. These findings show that FGF‐BP gene overexpression has a greater relative effect on wound healing in the diabetic rat model. The cDNA also had a significant effect in a rabbit excisional wound model that depended on granulation tissue formation. Truncated forms of the molecule may have higher therapeutic potency. FGF‐BP has an important role in FGF‐1/2 mobilization and macrophage functions, and FGF‐BP gene therapy for wound healing can improve the process by stimulating angiogenesis, epithelization and collagen synthesis in target tissue.
039 Determination of Burn Depth using near Infrared Spectroscopy
Authors: Cross K , Leonardi L , Fish J , Sowa M , Gomez M , Payette J , Hastings M
Published: March 2007

Introduction: Burn depth, based on the hemodynamic alterations that occur following a thermal insult, can be assessed in a rapid, non‐invasive, and nondestructive fashion using near infrared (NIR) spectroscopy. NIR has the capability to determine the difference between superficial and full thickness burn injuries.

Methods: Sixteen burn patients admitted to an adult regional burn center were studied and evaluated with the NIR point and imaging devices. Non‐burned skin adjacent to the burn site was used as the control. NIR measurements were compared between superficial (8 wounds), full thickness (8 wounds) burn wounds and control sites.

Results: NIR was able to easily detect an increase in oxyhemoglobin (68.3%, p < 0.05), oxygen saturation (4.8%, p < 0.05%) and total hemoglobin (91.3%, p < 0.05) which typically occurs with superficial burn injuries. Full thickness injuries experienced a substantial drop in oxyhemoglobin (88.8%, p < 0.05), oxygen saturation (79.1%, p < 0.05) and total hemoglobin (77.5%, p < 0.05) in comparison to control sites.

Conclusions: These results confirm that NIR spectroscopy can successfully distinguish between superficial and full thickness burn injuries. The second phase of this study will involve determining the depth of indeterminant burn wounds and this preliminary data will also be presented.

Acknowledgement: National Research Council of Canada
040 A Chronic Pressure Sore Nude Mouse Model
Authors: Wassermann E. , Gstaltner K. , Redl H.
Published: March 2007

The epidemiology of pressure sores is not clearly understood yet. Tissue layers involved in the pressure sores include skin, subcutaneous fat, deep fascia, muscle, and bone. Widely used experimental models lead to the pressure sores in the skin and the underlying fatty tissue only. The aim of the present study was to develop a clinically relevant deep pressure sore model in nude mice. METHODS: Six weeks old Balb/c nude mice weighing 25–30 g were used. After introduction of anesthesia a steel disk (Ø 5 mm, 0.5 mm thickness) was implanted under the musculus gluteus max.. The wound was closed with 4/0 sutures. The full recovery was allowed for 10 days. Animals with an insufficient healing were excluded. The pressure was induced using a Neodymium magnet (1.29 Tesla) in conjunction with the disk in un‐anesthetized mouse periodically. A two hours compression was followed by one hour recovery for 4, 6 or 8 cycles, mimicking clinical setting. The controls underwent the same procedure, including the disk implantation but without placement of the magnet. RESULTS: The pressure sore depth was found to be cycle dependent – with the highest degree after 8 cycles. The histologic evaluations revealed signs of necrosis in the skin and subcutaneous fat after 4 and in the muscle only after 8 cycle of compression. PMN infiltration into the skin, subcutaneous fat, the deep fascia, and muscle was found in all groups. CONCLUSION: This model with clinically relevant magnitude and duration of pressure application leads to an injury in all tissue layers similar to the patient setting. This nude mouse model can be used to investigate the mechanism of pressure sores development and to study new therapeutic approaches (e.g. human cells) without the need of immunosuppression.
042 FGF‐BP improves wound healing through the induction of more collagen in rats
Authors: Shi Y. , Liu J. , Bittner M. , Goppelt A. , Davidson J.M.
Published: March 2007

The fibroblast growth factors are important regulators during wound healing. To quench their biological activities, secreted FGFs are tightly bound to heparan sulfate proteoglycans in the extracellular matrix (ECM). One of the approaches for releasing the active FGF from ECM involves the binding to an FGF binding protein (FGF‐BP), which prevents FGF degradation and retains its activities. FGF‐BP enhances the proliferation of fibroblasts through FGF‐1 and ‐2 and of endothelial cells through FGF‐2. To detect the biological function of FGF‐BP in wound healing, we constructed an adenoviral vector containing murine FGF‐BP cDNA (Ad‐FGF‐BP). Polyvinyl alcohol (PVA) sponges were implanted subcutaneously in rats. 107–108 PFU of either Ad‐FGF‐BP or Ad‐LacZ (as local control) was injected into the sponges at d3 after implantation. At d4 after injection, Ad‐FGF‐BP infected sponges displayed much better organization of granulation tissue with the presence of more macroscopic hemorrhage than the local control. At d7 after injection, Ad‐FGF‐BP infected experimental granulation tissues in sponges showed more collagen deposition. The contents of collagen, protein and DNA in Ad‐FGF‐BP infected sponges increased 24.0%(p < 0.05), 11.3%(p < 0.05, d4) and 28.2%(p < 0.001, d7), respectively. Incisional wounding was performed in rats and 108 PFU Ad‐FGF‐BP or Ad‐lacZ was intracutaneously injected along the wound margins. At 7d after injection, the tensile strength of Ad‐FGF‐BP infected wounds increased 41.1%(p < 0.05). Our data indicate that overexpression of FGF‐BP can improve wound healing, and the inductionof more collagen in granulation tissue may be one of the mechanisms.
043 Iκ‐B Adenoviral Gene Transfer Improves Wound Healing
Authors: Schreiber J , Efron P , Park J , Moldawer L , Barbul A
Published: March 2007

Introduction: One of the earliest events in wound healing and scar formation is the inflammatory phase. Deficient or excessive inflammatory responses result in aberrant healing. NFK‐b is a transcription factor that regulates key genes responsible for multiple intra‐nuclear inflammatory cytokines. When bound to IK‐B, an inhibitory protein, this factor is in an inactive state. We studied the wound healing effects of irreversibly inhibiting the activation of NFK‐b by continuously infusing adenoviral activated IK‐B.

Methods: Seventy‐five male Sprague‐Dawley rats underwent subcutaneous implantation of polyvinyl alcohol sponge‐osmotic pump constructs. The pumps were filled with a solution containing a genetically altered adenovirus containing the DNA for continuously activated IK‐B protein. 50 μl of viral transport media containing 5 × 109 PFU of virus were delivered to each individual sponges daily. Controls included pumps filled with normal saline in one group or filled with sham virus in another group. Sponges were harvested on days 1,3,5, and 7 post‐implantation. The sponges were analyzed for TNF‐α and nitric oxide (NO) levels, as index of inflammation, as well as for hydroxyproline (OHP) content at seven days, an index of collagen deposition.

Results: Treatment with the IK‐B virus resulted in significantly higher levels of OHP after seven days when compared to normal saline and sham virus treated sponges (654 ± 81 vs 546 ± 109 vs 498 ± 123 μg OHP/100 mg sponge, P < 0.05 by ANOVA). Wound fluid NO concentration was significantly lower in the IkB group after 5 days (38 ± 15 vs 55 ± 12 vs 71 ± 27 μM, P < 0.05 by chi‐square). There were no differences in sponge TNF‐α concentrations on days 1,3, and 5.

Conclusions: Treatment of wound sponge granulomas with IK‐B increases the amount of collagen deposition after seven days. Reduction of inflammation by inhibiting NF‐KB may be a possible mechanism of action, as reflected in decreased NO wound content.
047 rAd‐p21 Significantly Attenuates Granulation Tissue Formation and Scar Thickness In Vivo
Authors: Gu D‐L. , Atencio I. , Looper D. , Kang D.W. , Maneval D. , Zepeda M.
Published: March 2007

Excessive scarring is a significant clinical problem. p21, a cyclin‐dependent kinase inhibitor, blocks cell cycle progression and attenuates proliferation of various cell types. Our in vitro rAd‐p21 dose response studies of primary human dermal fibroblasts showed a 3–80 fold reduction of cell proliferation as measured by BrdU incorporation at doses of 1 × 108−3 × 109 PN/mL, respectively. Further, rAd‐p21 at 3 × 109 PN/mL showed a 2‐fold less procollagen I (PIP) production when compared to control virus. These in vitro data demonstrate that rAd‐p21 significantly attenuates fibroproliferation and procollagen production in the target cells. In vivo, a rat PVA sponge model was used. rAd‐p21, at doses of 1 × 109, 1 × 1010 and 5 × 1010 PN was delivered into sponges that had previously been treated with a granulation tissue stimulator, rAd‐PDGF‐B. Results showed that sponges receiving rAd‐PDGF‐B alone had the highest % granulation fill (40–60% fill area). Sponges that were treated with rAd‐PDGF‐B for the first injection followed by rAd‐p21 on a second injection showed a dose‐dependent decrease in % granulation fill as rAd‐p21 doses increased (p < 0.001). On day 5 post‐injection, IHC staining identified p21 protein expression only in sponges receiving rAd‐p21. In addition, the quantitative measurement of cells labeled by BrdU or Ki67 demonstrated that rAd‐p21 significantly attenuated cell proliferation when compared to rAd‐PDGF alone in this model (p < 0.01). In another in vivo experiment, a rabbit excessive scar model was used. rAd‐p21, at a dose of 2 × 109 PN/wound was intradermally injected into the rabbit ear wounds that had previously been treated with PDGF‐BB protein. Preliminary data showed that scar thickness in rAd‐p21 treated wounds was significantly decreased in comparison to wounds treated with the control virus (p < 0.05). These data suggest that rAd‐p21 is effective in attenuating scars in the rabbit ear excessive scar model. Our in vitro and in vivo data support the further exploration of rAd‐p21 as a useful therapeutic candidate for hyperproliferative diseases in patients.
049 Lactate Controls Vascular Development in Wound Healing
Authors: Aslam R. S. , Scheuenstuhl H. , Beckert S. , Hopf H. , Hussain Z. , Hunt T. K.
Published: March 2007

INTRODUCTION: High concentrations of lactate approx. 10 mM are characteristic of wound healing. We have previously shown that lactate instigates release of VEGF from macrophages and endothelial cells. Our present study uses an in vivo murine Matrigel model to study how lactate enhances vascular development and repair.

METHODS: Matrigel is a reconstituted basement membrane complex, a liquid at 4 degree C. When injected subcutaneously, it reconstitutes as a gel. 30 mgs of finely divided polylactate polymer (DL‐lactide‐co‐glycolide) was mixed with 1cc of Matrigel, and 2 injections were made into the dorsum of each of 60 (average 7/group), 6‐month‐old Swiss Webster mice. The gel implants were harvested at 3, 6, 9, 11 and 16 days post injection, and were fixed in buffered formalin or frozen. Cell migration and vascular development were assessed using H&E, anti‐CD31 and anti‐MAC3 antibody stainings for leukocytes, fibroblasts, endothelial cells, and macrophages. Cells and vessels were quantified microscopically.

RESULTS: Control implants (without polylactate) developed little or no inflammation or angiogenesis. Polylactated implants developed highly significant number of endothelial cells and recognizable vessels at 6 and 9 days. At 11 and 16 days, sizeable vessels with surrounding connective tissue were found. Though a moderate inflammation occurred in the lactate groups, no foreign body reaction was seen. Larger implants of high molecular weight instigated little inflammation and few vessels.

CONCLUSION: Slowly released lactate leads to the formation of new vessels. Our results provide support for further clinical applications of lactate polymer in wounds.

NIH/NIGMS Trauma Training Grant
050 The Angiogenesis Inhibitor Endostatin Impairs Wound Healing at Tumor‐inhibiting Doses
Authors: Michaels J , Dobryansky M , Galiano RD , Bhatt KA , Ashinoff R , Ceradini DJ , Levine JP , Gurnter GC
Published: March 2007

Introduction: Angiogenesis is thought to be important for granulation tissue formation and for the delivery oxygen and other nutrients to the healing wound bed. We examined whether angiogenesis inhibitors (AI) such as endostatin decrease neovascularization in full‐thickness wounds leading to impaired granulation tissue formation and delayed wound closure.

Methods: Endostatin at tumor‐inhibiting doses (20 mg/kg/BID) was injected daily starting three days prior to surgery. Two full‐thickness wounds were created on the mouse dorsum using a novel wound healing model developed in our lab. A second experimental group had topical VEGF (10 μg/QD) applied to these wounds. Both groups were compared to PBS‐treated controls. Wounds were analyzed for closure time, granulation tissue formation, and wound vascularity using CD31.

Results: Endostatin‐treatment delayed wound closure compared to control mice (17.4d ± 1.51 vs. 12.8 ± 0.89, P < 0.05), resulted in decreased granulation tissue formation at all time points (P < 0.05), and significantly reduced wound vascularity as measured by CD31+ vessel counts (P < 0.05). VEGF application to the wound bed of endostatin‐treated mice normalized wound closure despite endostatin treatment (13.8d ± 1.1 vs. 17.44d ± 1.51, P < 0.05).

Conclusion: Endostatin impairs wound angiogenesis, granulation tissue formation and delays full‐thickness wound closure. Topical VEGF was able to reverse this effect and may represent a novel approach to improve wound healing in patients receiving AI. These findings may have serious implications for patients undergoing AI treatment that require surgery or who have wound healing complications.

Acknowledgements: This project received no outside funding.
052 Redox Control of Dermal Wound Healing
Authors: Sen Chandan K. , Pendyala Srikanth , Khanna Savita , Hunt Thomas K. , Roy Sashwati
Published: March 2007

Reactive oxygen species (ROS) are generally thought to be deleterious to biologic systems. The phagocyte NADPH oxidase provides a classical example of deliberate ROS generation. High amounts of ROS are generated in transient bursts to kill pathogens. The recent discovery of another family of NADPH oxidases, the Nox/Duox family, provides additional examples of deliberate generation of ROS by non‐phagocytic cells present at the wound site. Our working hypothesis is that ROS generated by wound‐related cells (low‐ROS by Nox; and residual ROS in the aftermath of phagocyte respiratory burst) support early‐phase acute wound healing by inducing redox‐sensitive signal transduction pathways especially those related to wound angiogenesis.

We have observed that ROS‐sensitive redox signaling pathways drive wound angiogenesis. Genetic as well as pharmacological approaches to deliver low concentrations of ROS promote dermal wound angiogenesis, contraction and closure. While at such low levels ROS do not serve as a potent disinfectant, they may act as modulators of cellular response facilitating the healing process. Strategies to decompose ROS at the wound site impaired healing. Congenital defect in human NADPH oxidase results in impaired wound healing as well as increased susceptibility to infection. We have observed that NADPH oxidase deficient transgenic mice suffer from impaired healing even under super sterile conditions; the impairment is corrected by low‐dose ROS delivery. Dermal wound healing is subject to tight redox control. The state of wound tissue oxygenation is a key factor that enables such control.
056 In Vitro Evaluation of Biofilm Development on Tissue Engineered Skin
Authors: Charles CA. , Ricotti CA , Davis SC , Kirsner RS
Published: March 2007

Bacteria reside in various forms, including planktonic, or free‐floating, or as biofilms, which are a tightly adherent, potentially more treatment‐resistant form. Biofilms, composed in part as polysaccharide matrices, can act as protection for bacteria by shielding them from antimicrobials and the innate immune system. It is estimated that bacterial biofilm are responsible for 65–80% of all chronic infections and may be responsible in part for non‐healing of chronic wounds.

An advance in the treatment of non‐healing wounds has lead to the development of human skin equivalents (HSE), which can successfully treat many non‐healing wounds. Better understanding the interaction between bacteria and HSE might improve patient outcomes. Studies involving planktonic E. coli and HSE have demonstrated the growth of bacterial colonies only on the dermal surface. Growth was not seen on the epidermal layer of the HSE, possibly due in part to the expression of innate antimicrobial peptides called human β defensin‐2 (hBD‐2), which are expressed by HSE keratinocytes. The objective of this study was to determine if biofilm formation could occur on a bioengineered HSE.

We used a commercially available HSE. Three (3) mm full‐thickness incisions were made in a triangular section of HSE. Each section was inoculated on the epidermal aspect with 1.0 × 105CFU per gram of Pseudomonas aeruginosa at 35 degrees Celsius for specified time points. Sections of HSE were sampled in areas of injury at various time points. Biopsy sections were processed for histologic analysis with H&E and epifluorescent microscopy to visualize Pseudomonas aeruginosa. We found that biofilm formation occurred at multiple time points on HSE. Colonies of adherent bacteria were visualized within the injured area of the epidermal layer of HSE by H & E staining. Eplifluoresecnt microscopy using calcofluor white staining revealed the characteristic exopolysaccharide (EPS) matrix of biofilm.

Visualization of the expression of human β defensin‐2 (hBD‐2) with HSE after biofilm formation is underway. This knowledge will help to understand the role of bacterial biofilms within HSE and their effect on subsequent healing.
057 Epifluorescent and light microscopic visualization of bacterial microcolonies in acute wound infections – Are these biofilms?
Authors: Ricotti Carlos , Cazzaniga Alex , Chen Halland , Mertz Patricia M. , Davis Stephen
Published: March 2007

A biofilm is a formation of surface‐associated microbial cells that are enclosed in a self produced extracellular polymeric substance matrix. This definition is acceptable for in‐vitro research but a clear definition for biofilm‐associated diseases has yet to be elucidated. A structural/morphological definition is currently used to define biofilms; additionally, physiological or molecular criteria will allow us to define biofilms in disease infection accurately. Our objective over the past several years has been to characterize and observe bacterial biofilms in wound infections using different wound models. To broaden our current understanding of biofilm morphology in wounds for this study we used epifluorescent and light microscopy to visualize wound pathogenic pseudomonas aeruginosa bacteria in a porcine partial thickness infection model.

Three experimental animals were used for this study. After animal preparation, partial thickness wounds were created on the backs of 3 animals. Wounds were then inoculated with 106 colony forming units/ml (CFU/ml) of Pseudomonas aeruginosa and covered for 48 hours to allow bacteria to colonize and infect the wound. Biopsies were obtained from normal skin, wounds before inoculation, wounds at 48 hours and wounds 48 hours after inoculation. Biopsies were processed and stained with Hematoxylin and Eosin for light microscopy and Calcofluor White and Ethidium Bromide for epifluorescence microscopy.

Images obtained using epifluorescent and light microscopy demonstrate that bacteria form aggregates of microcolonies. These structures are representative of bacterial biofilms and support the hypothesis that bacteria live as biofilms in wound infection. Although currently there is no established and accepted definition for biofilm associated diseases, we anticipate that more studies looking into physiological changes of these structures will clarify our current understanding of wound infection and treatment.
058 Silver Dressings: Effective Antimicrobial Therapies?
Authors: Davis SC , Ricotti C , Cazzaniga AL , Mertz PM
Published: March 2007

Many silver‐dressings have been recently developed to prevent infection of burn and chronic wounds. Antimicrobial therapies are usually studied in in‐vitro assays, which evaluate the antimicrobial efficacy against free‐floating bacteria. In a wound environment bacteria attach to tissues and establish bacterial biofilms. Biofilms are communities of colonies of bacteria and other microorganisms encased in a self produced exopolymeric substance. Biofilms help protect the bacteria from the environment and limit the effectiveness of antimicrobials. In this study we evaluated the efficacy of two silver dressings to eradicate biofilm‐associated and planktonic Pseudomonas aeruginosa cells using our biofilm burn wound animal model.

Three pigs were used in this study. Second‐degree burn wounds were made on one‐half of the animal’s back and inoculated with a burn wound isolate of P. aeruginosa. These wounds were then covered for 72 hours with a polyurethane dressing to allow biofilm formation. After 72 hours, additional burn wounds were made on the unwounded half of the animal. The new burns were then inoculated with the same P. aeruginosa strain. At this 72 hour point we had established two bacterial groups, one side of the animal had burns with planktonic bacteria and the other half with biofilm bacteria.

Both sides of the animal were treated 20 minutes after inoculation of the planktonic group with the following dressings: 1) Nanocrystalline Silver, 2) Hydrocolloid Silver, or 3) untreated. Wounds were cultured from all treatment groups at 24, 48 and 72 hours post treatment. Sites were cultured quantitatively using a novel flush‐scrub technique to obtain both planktonic and biofilm bacterial counts. The baseline biofilm bacterial count was 7.56 LogCFU/ml (prior to treatment).

The hydrocolloid dressing significantly reduced planktonic bacteria counts as compared to untreated and nanocrystalline silver dressing at 24, 48 and 72 hours. However, biofilm bacteria counts for both dressings were similar to untreated control at all sample points. Our study demonstrates that both silver dressings showed limited effect against P. aeruginosa biofilm‐associated cells when compared to untreated wounds.

Based on our results we question the effectiveness of silver dressings for infected wounds that are colonized with biofilm associated‐cells. We conclude that anti‐biofilm susceptibility models may improve on current antimicrobial sensitivity assays.
061 Predictors of Poor Outcome in S. Aureus Wound Infection
Authors: Tibor L. , Cramton S.E. , Hopf H.W. , Götz F. , Hunt T.K.
Published: March 2007

Objectives: Staphylococcus aureus is the most common bacterium cultured from infected wounds. We hypothesized that diabetes mellitus (DM) and/or peripheral arterial disease would be patient predictors of severe outcomes, defined as amputation, sepsis, or death, and infections caused by resistant strains would be bacterial predictors of severe outcomes.

Methods: With IRB approval, 50 S. aureus strains (45 wound, 5 central line) were isolated during routine clinical care. Investigations included antibiotic resistance (>5 mcg/ml), and PCR for STAR elements. Chart review included wound type, location, relevant comorbidities, antibiotic resistance, and outcome.

Results:

Outcome n(%of outcome total)

Severe
All others
Total

Diabetic
7 (70)
6 (17)
13 (29)

Non‐diabetic
3 (30)
29 (83)
32 (71)

MRSA STAR groups
2 (20)
8 (23)
10 (22)

All other STAR
8 (80)
27 (77)
35 (78)

MRSA
4 (40)
8 (23)
12 (27)

MSSA
6 (60)
27 (77)
33 (73)

Total patients
10 (22)
35 (78)
45

No non‐DM patient with arterial disease (n = 6) had a poor outcome.

Conclusions: The strongest predictor of outcome was the presence of DM. Patients with severe outcomes were slightly more likely to have MRSA infection, although only 33% of patients with MRSA had severe outcomes. Although STAR/arsRBC groups have been shown previously to predict antibiotic resistance, in this collection there was no correlation between these molecular markers and outcome. This study shows that the strongest predictor of clinical outcome in wound infection is patient‐dependent, while bacterial factors appear to play a lesser role.
062 The Impact of Nosocomial Resistant Pseudomonas Infections in a Burn Unit
Authors: Tredget Edward E. , Shankowsky Heather A. , Lee Jonathan P. , Swanson Todd
Published: March 2007

Introduction: Pseudomonas aeruginosa, remains a serious cause of nosocomial infection and septic mortality in burn patients particularly when nosocomially acquired. Our purpose is to investigate the morbidity and mortality associated with nosocomial infection with an aminoglycoside resistant Pseudomonas and associated costs compared to a group of patients with similar severity of burn injury that did not acquire resistant Pseudomonas during hospitalization.

Methods: Using a TRACS burn database, patients treated at our institution with Pseudomonas resistant to gentamicin were identified and case‐matched to controls for age (±5 years), TBSA(±5%), admission year (±5 years) and presence of inhalation injury. Patients who died <48 hours after injury were excluded. Data examined included demographics, number of days to onset of positive Pseudomonas culture and gentamicin resistance, as well as antibiotic use and cost.

Results: 42 patients admitted to our unit between 1980 and 2001 were identified with gentamicin resistant Pseudomonas. Patients in the resistant Pseudomonas(Ps) group were similar in age (39.2 ± 3.2 vs 40.4 ± 3.3 years), TBSA (47.5 ± 3.7 vs 48.9 ± 3.7%), extent of full thickness injury (37.0 ± 3.9 vs 30.3 ± 3.2%) and presence of inhalation injury (62.8% vs 55.0%) compared to controls. There was a significant increase in the mortality rate in the Ps group (39.5 vs 5.0%, p < 0.001)(paired t test) compared to controls and the morbidity in terms of length of stay, increased in the Ps group (73.1 ± 13.2 vs 55.8 ± 8.1 days). Ventilatory days (22.6 ± 5.1 vs 8.2 ± 2.4, p < 0.05), number of surgical procedures (4.5 ± 0.6 vs 2.9 ± 2.5, p < 0.05), and amount of blood products used (packed cells 47.9 ± 7.8 vs 18.6 ± 3.3, p < 0.01)(platelets 10.5 ± 2.9 vs 0.5 ± 0.3, p < 0.01) were all significantly higher in the Ps group compared to control. Costs associated with antibiotic requirements were also significantly higher in the Ps group ($3,191.90 ± 848.00 vs $613.60 ± 145.50, p < 0.01).

Conclusions: Our data demonstrate that nosocomial infection in burn patients with aminoglycoside‐resistant Pseudomonas is associated with significantly higher morbidity and mortality and cost of care. Increased resource consumption in terms of length of stay, number of surgical procedures, amount of blood products, and antibiotic costs did not prevent significantly higher mortality rates when compared to control patients who avoided infection with the resistant organism. Thus, prevention, identification and eradication of nosocomial Pseudomonas infection are critical for cost‐effective, successful burn care.
065 Improving Integra™“Take” with Negatively Charged Methacrlylic Acid Beads
Authors: Eckhaus AA , Fish JS , Sefton MV
Published: March 2007

Introduction: Integra™ has gained acceptance as a dermal replacement for large burns with limited donor sites. Integra™ vascularization takes between 14 and 21 days. Accelerating the vascularization of Integra™ could improve “take” rates and potentially decrease the obligatory waiting period between application and autografting.

Hypothesis: Application of negatively charged methacrylic acid (MAA) beads will enhance vascularization of Integra™

Methods Male Wistar rats (n = 11) had two 2 × 3 cm contra lateral dorsal full thickness wounds excised and grafted using Integra™. The MAA beads were applied under the Integra™ topically on the wound bed. Three experimental treatments were compared: Group 1‐ high‐dose MAA beads, Group 2‐ low‐dose MAA beads, Group 3‐ Integra only. Laser Doppler Imaging (LDI) to assess perfusion, as well as digital photography was done on days 7,10 and 14. Tissue samples for H + E and Factor VIII histological staining were collected on day 14.

Results: The average “take” was 99%(p < 0.05) for high‐dose MAA beads, 98%(p < 0.05) for low‐dose MAA beads and 82% in the Integra™ only group at day 7. At day 7 (p < 0.01) and day 10 (p < 0.05), the low‐dose MAA group had significantly greater perfusion than the Integra™ only group. There were no statistically significant differences at day 14. Microvessel density (MVD) counts revealed a >40% increase in the number of vessels in both the low‐dose MAA (p < 0.05) and high‐dose MAA (p < 0.05) groups when compared to the Integra™ only group. There was no difference in LDI perfusion or MVD counts between low and high‐dose MAA groups.

Conclusion: Factor VIII staining revealed enhanced angiogenesis in Integra™ treated with low and high‐dose negatively charged MAA beads. Low‐dose negatively charged MAA beads improved and accelerated the vascularization of Integra™ in this rodent model.
068 Photoresponsive Hydrogel Scaffolds in Cutaneous Repair
Authors: Andreopoulos F.M. , Persaud I. , Gattas‐Asfura K.M. , Leblanc R.M.
Published: March 2007

Bioactive scaffolds impregnated with multiple growth factors that are released at controlled rates, offer the possibility of enhancing the healing process by mimicking the work of the natural tissue. We have developed photoresponsive and biodegradable hydrogel matrices based on natural and synthetic polymers that can undergo phase transformations upon exposure to alternating wavelengths of irradiation. Photo‐polymerization provides an effective and benign method for in situ hydrogelation with spatial and temporal control of the polymerization reaction. Polyethylene glycol, heparin and gelatin molecules have been modified at high yields with photosensitive groups that can be rapidly and reversibly photocrosslinked to hydrogel scaffolds in the absence of initiators. We are currently exploring the efficacy of these scaffolds as depots for the controlled delivery of basic fibroblast growth factor in cutaneous regeneration. We have demonstrated that the stability of b‐FGF is enhanced by its incorporation within the gel matrix and its complexation with components of the scaffold (i.e. heparin) while the releasing peptide maintains its activity. Cell proliferation and partial thickness wound studies demonstrated that b‐FGF could be affectively released from the scaffolds at controlled rates and its wound healing properties were a function of the mode of delivery. Rate changes up to 60% on the kinetics of peptide release could be easily achieved by altering the exposure times (seconds to minutes range). Similarly, we were able to alter the release profile of b‐FGF by adjusting the ratio of the photosensitive polymeric macromers prior to crosslinking. These results suggest that the proposed photoresponsive gels scaffolds could serve as bioactive peptide delivery vehicles in a number of tissue engineering applications.
069 Scar Reduction by Adhesive Patches: Efficacy and Potential Mode of Action
Authors: Meyer‐Ingold W. , Achterberg V.
Published: March 2007

There is a wide spectrum of treatment modalities to manage hypertrophic scars, techniques like surgical excision, intralesional application of corticosteroids, pressure therapy or laser therapy are extensively used.

From a cosmetic and physiological point of view scars are predominantly a concern of the patients, the cosmetic relevance for doctors is often of less importance. Therefore a treatment that can be performed by the patient her‐/himself might be advantageous if it is efficacious and safe. Since the early 1980s so‐called “silicone gel sheeting” has been a widely used safe clinical management option for hypertrophic scars and keloids.

The principle of the latter treatment has now been adapted and optimised towards a specific easily applicable adhesive polyurethane patch. This breathable, hypoallergenic patch is very well tolerated and avoids – in contrast to silicone sheets – moisture accumulation on the skin. Several clinical studies on both mature scars and on scar formation after surgical incisions show a reduction in visibility, redness, and roughness of the scars. The recommended eight weeks treatment might even be interrupted daily for twelve hours; the scar reducing effect is achieved as well.

Extensive investigations on the polyurethane patch in vivo and in vitro propose an improved remodelling process of the scar tissue due to thermal and pressure effects of the patch but not by stratum corneum hydration. In vivo a better microcirculation can be observed in the scar tissue after treatment and in vitro an activation of enzymes involved in the remodelling process can be measured.

In summary, physical effects are likely to be responsible for the successful results in clinical studies and individual applications of the new polyurethane patch for the reduction of hypertrophic scars.
071 TGF‐ß‐Mediated Paracrine Regulation of Keratinocyte Wound Closure
Authors: Sharif‐Afshar A. , Gorska A. E. , Neilson E. G. , Davidson J. M. , Moses H. L. , Bhowmick N. A.
Published: March 2007

The importance of stromal‐epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF‐ß family. TGF‐ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF‐ß type II and type I receptors, respectively. We address the role of TGF‐ß signaling in dermal fibroblasts using a conditional fibroblastic TGF‐ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF‐ß signaling in the dermal fibroblasts results in accelerated excision‐wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re‐epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast‐conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno‐histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF‐ß signaling by the dermal fibroblasts suppresses re‐epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms.   Funding: DOD BC99184 and NIH CA85492.
074 Wound Healing Enhancement by Pulsed Electromagnetic Fields
Authors: Callaghan M.J. , Kinnuncan E.R. , Michaels J. , Simon B.J. , Gurtner G.C.
Published: March 2007

The effects of pulsed electromagnetic fields (PEMF) on soft tissue are not well characterized. This study utilized a diabetic wound model to examine the effects of PEMF effects on soft tissue healing.

Methods: Wounds were created on the dorsum of db/db and wild type C57BL6 mice. Mice were exposed to PEMF (4.5 ms pulse/15 hz) for 8 hrs/day for 14 days. Gross closure was assessed with digital analysis of area changes over time. Histological examination assessed granulation and epithelial gap, cell proliferation (BrdU), and endothelial cell density (CD31). Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of PEMF for 8 hrs and VEGF/FGF2 was measured in culture supernatants by ELISA.

Results: Mice exposed to PEMF had accelerated wound closure at day 7 (wound area as % of original, db/db: 60%(PEMF) vs. 78%(control), C57BL6: 15% vs. 42%, p < 0.05) and day 14 (db/db: 21% vs. 55%, C57BL6: 8% vs. 28%, p < 0.05), with increased granulation and cell proliferation (db/db day 7: 52 ± 8 vs. 31 ± 5 cells/HPF (200x)). Immunohistochemical analysis revealed significantly higher CD31 density in wounds exposed to PEMF at day 7 (vessels/HPF, db/db: 28 ± 4 vs. 17 ± 4, C57BL6: 41 ± 7 vs. 28 ± 6) and day 14 (db/db: 32 ± 6 vs. 21 ± 5, C57BL6: 48 ± 5 vs. 40 ± 5). HUVECs in PEMF exhibited 5‐fold higher levels of FGF2 compared to controls after 30 min (20.50 pg/ml ± 6.75 vs. 4.25 pg/ml ± 0.75), with no change in VEGF through 8 hrs.

Conclusions: PEMF accelerates closure time and endothelial cell proliferation in wound healing. Upregulation of FGF2 in HUVECs exposed to PEMF suggests that release of angiogenic growth factors may explain increased vascular density and accelerated wound closure. Clinical uses include treatment for diabetic ulcers and other non‐healing wounds.

This work is supported in part by the Alumni Fund, Alumni Association of SUNY Downstate College of Medicine (MJC).
082 CM‐1 (Cytomodulin), a Synthetic peptide, promotes collagen transcription and wound healing in bioluminescent mice
Authors: Dasgupta Jayasri , Bhatnagar Rajendra , Davidson Jeffrey M
Published: March 2007

Cytomodulin (CM‐1) is a synthetic heptapeptide. Human dermal fibroblasts cultured in the presence of CM‐1 showed increased expression of transforming growth factor–ß(TGF‐ß) and collagen I and decreased expression of MMP‐1. TGF‐β is involved in numerous vital processes in wound healing, and the rate of wound repair is influenced by its overexpression at the site of injury. To establish the effect of CM‐1 in wound healing, we administered several concentrations of CM‐1 to incisional wounds and to unwounded skin of collagen‐luciferase transgenic mice. Luciferase bioluminescence served as a quantitative reporter of CO1A2 transcription. Imaging was performed to measure the luciferase activity on these mice at different time points after administration of 1, 10 and 100 ug CM‐1. The highest amount (100 μg) showed the highest level of collagen expression both in unwounded and wounded skin. In another experiment, we used a scrambled peptide as a control and administered the peptides both topically and by injection. CM‐1 was effective in stimulating luciferase expression both by topical and intradermal administration. To determine the effect of CM‐1 on wound strength, we measured the tensile strength in incisional wounds of wild type C57B6 mice at 10 d. Histological analysis was also performed to assess the collagen content and organization of the tissue. The data showed a statistically significant increase in tensile strength and collagen content at 10 and 100 μg doses. These results illustrate the utility of CM‐1 as a novel, low‐molecular weight vulnerary agent.