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Notes: 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.

Notes: The complexity of cellular and molecular events that occur during wound healing is given by the rapid and continuous changes of matrix structure, cell population, local cytokine composition, nutrient and oxygen availabilities and overall metabolic activity. Angiogenesis is an essential event in the repair of injured tissue. Transcriptional events leading to wound neovascularization begin immediately after the injury has occurred and continue throughout the process until scar remodeling becomes quiescent. This study investigated progression of wound angiogenesis by quantitatively measuring transcripts of multiple genes that participate in wound angiogenesis. The studies were done using a murine model of bilateral 3 mm excision wounds. Wound tissues were collected from 6 hours up to 10 days post-wounding. Each time point consisted of 8 wounds pooled from four different mice. After RNA purification, the samples were analyzed by quantitative real-time RT-PCR using specific TaqMan probe/primer sets for selected transcripts that included the following groups: a) angiogenic factors; b) anti-angiogenic factors; c) receptors; d) cytokines and enzymes; e) matrix proteins; f) signaling molecules and transcription factors; g) apoptosis and proliferation markers; and h) housekeeping genes. A total of 58 genes were profiled including four housekeeping genes. The transcripts revealed several patterns of expression. Some genes such as angiopoietin 1(Ang1), e-selecting, placental growth factor (PlGF), MMP-9 and thrombospondin 1(TSP1) had a clear biphasic pattern where early peaks were detected within the first 24 hours after wounding and were followed by dowregulation and a second increase in transcription after several days. Other transcripts appeared early and had a sustained expression and they include fibroblast growth factor two (FGF2), FGF7, hypoxia inducile factor alpha (HIF-1a) and STAT3. Examples of late transcripts included HOXD3 and alpha smooth muscle actin. Results derived from this study largely confirm previous studies profiling a small number of individual genes such as VEGF, TSP1 and 2 and others. However, the present study integrates complete transcriptional profiles of large number of genes known to have a critical role in healing progression with a particular emphasis on revascularization. A more detailed model of event sequences that lead to tissue reperfusion after injury is derived from the results.Funded by a Cedars-Sinai grant from the Skirball-Kenis Center for Plastic and Reconstructive Surgery and RO1GM066292 to MRSH.

Notes: 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.

Notes: Introduction: Ischemia-reperfusion (IR) injury is commonly associated with numerous pathologies including pressure sores, venous stasis ulcers, and lower extremity diabetic ulcers. The purpose of this study is to further investigate the relationship between age and ischemia-reperfusion skin injury in a rat model utilizing magnets for the purpose of injury creation.
Methods: Magnets were designed for subcutaneous placement and calibrated such that a second magnet placed externally over them would cause compression that exceeds capillary perfusion pressure (ischemia). Removing the external magnet results in reperfusion of the skin. After placing subcutaneous magnets in aged and young Fisher 344 rats, repeated cycles of external magnet placement and removal were performed. 
Results: Visual analysis of the skin revealed statistically significant greater areas of injury in the aged rats relative to their younger counterparts (37.4 ± 13.3% vs. 24.1 ± 14.8%, P 〈 .02)
Conclusions: Aged rats demonstrate an increased degree of injury relative to their younger counterparts in response to ischemia-reperfusion injury. Future studies will attempt to delineate differences in the markers of IR injury (such as myeloperoxidase and vitamin E levels) in aged versus young rats, giving insight to the mechanisms responsible for the impaired wound healing seen in the elderly.

Notes: Introduction: Telomeres are nucleoprotein structures at the ends of each chromosome. Due to the inability of DNA polymerase to replicate the full length of the chromosome, up to 50–200 base pairs of the telomere are lost during each successive round of cell division. In adult human somatic cells, telomerase is not active resulting in progressive loss of telomere length and entry into replicative senescence as observed in cell culture. hTERT is the catalytic subunit of telomerase, an enzyme which maintains telomere length. Transfection of human dermal fibroblasts (HDFs) by hTERT has been shown to reverse the senescent phenotype seen in aging HDFs in vitro. ERK (p44/42) is a MAP kinase which functions as a critical intermediary in the determination of cell growth and differentiation. Activation of ERK occurs through phosphorylation of threonine and tyrosine residues. 
Methods: In order to delineate some of the cellular mechanisms by which hTERT functions, we treated adenoviral hTERT (Ad-hTERT) transfected HDFs with TGFB1, and assayed phosphorylated ERK activity by Western blotting.
Results: Ad-hTERT treated HDFs demonstrated a 2–3 fold increase in phospho-ERK activity. In addition, our preliminary findings show that Ad-hTERT transfected HDFs have increased TGFB1, TGFB1-Receptor I and II, and COL1A1 gene expression by real-time rtPCR. 
Conclusions: Increased phosphor-ERK activity as well as increased TGFB1, TGFB1-Receptor I and II, and COL1A1 gene expression is seen in hTERT transfected HDF’s. Further studies will focus on defining other intermediary changes resulting from Ad-hTERT tranfection.Funding source: Geron Corporation

Notes: 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.

Notes: In order to closely mimic the actual mechanism of wound debridement by an enzymatic debriding agent, a synthetic wound eschar substrate (SWES) has been developed. The SWES is prepared by the enzymatic conversion of fibrinogen to fibrin in the presence of fibrous bovine collagen and elastin, to form an insoluble planar matrix composed of the three major proteins present in wound eschar. Each of the proteinaceous components is uniquely labeled prior to formation of SWES with different dye, and the debridement test is conducted using a Franz Diffusion System. The debriding agent is applied to the SWES mounted on the donor compartment of the cell, and the digested hydrolysate containing dye-labeled peptide fragments is sampled from the receptor compartment. Accordingly, the progress of digestion is obtained in real time. Since the three proteins are labeled with spectrally distinct dyes, data on the hydrolysis of the substrates can be collected simultaneously. This method facilitates the testing of formulated debriding products in a manner that closely mimics application to a wound and provides a powerful tool for the evaluation of formulated enzymatic debriding agents.

Notes: 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.

Notes: Purpose: To evaluate expression patterns of protein kinases and protein kinase activities in human corneal fibroblasts treated with connective tissue growth factor (CTGF). Methods: Human corneal fibroblast cultures were grown to confluence and treated with CTGF for 0, 5, and 15 minutes. Cytoplasmic protein extracts were obtained; protein kinase expression and activity arrays were performed using Kinetworks™ analysis (screening for expression of 75 different protein kinases and 31 different phosphoproteins). Further studies using extended time courses of CTGF exposure in corneal fibroblasts (0, 1, 2, 3, 4, 5, 10, 15, 30, and 60 minutes) were performed using immunoblot analysis to detect expression of protein kinase A catalytic subunit (PKA-cat), and focal adhesion kinase (FAK). All results and were normalized by comparison to beta-actin. Results: After 5 minutes of exposure to CTGF, levels of active proteins increased for 21 of the 75 kinases analyzed. Some notable protein kinases that were induced include: death-associated kinase 1, focal adhesion kinase (FAK), G-protein coupled receptor kinase 2, protein kinase A catalytic subunit (PKA-cat), protein kinase B alpha, and protein kinase C (ε, μ, and ζ, subunits). Extended time course analysis of PKA-cat and FAK showed statistically significant increases in expression following CTGF stimulation within 15 minutes. Conclusion: CTGF increased the levels of active protein kinases in human corneal fibroblast cultures, including PKA-cat and FAK after 5 minutes of exposure. These results further our understanding of the signal transduction mechanism activated by CTGF in corneal fibroblasts. This suggests that CTGF mediates the effects of transforming growth factor beta (TGF-β) on protein kinase expression and phosphorylation of second messengers in processes such as cell proliferation and collagen synthesis. Acknowledgments: Funding source: NEI 05587

Notes: A critical limitation of using retroviral vectors for gene therapy is their inability to infect non-dividing cells. Although, the adenoviral vectors have the advantage of being able to infect both dividing and non-dividing cells, they elicit inflammatory response, thus making the interpretation of in vivo experiments harder. Adeno-associated virus (AAV) and Lentiviral vectors do not have those limitations, however, scant information is available about their transfection efficiency under low-oxygen tension. To determine if low-oxygen microenvironment affects viral vector-mediated gene transfection, we have used two other viral vectors, Adeno-associated virus (AAV) and Lentiviral constructs in vitro and in vivo to express foreign genes in hypoxic cultured human dermal fibroblasts and ischemic rat wounds. Both cultured normoxic and hypoxic (1% O2) human dermal fibroblasts were identically transfected by the AAV vector. A lenti6-LacZ construct was injected onto the periphery of rat ischemic and non-ischemic wound (106 pfu/wound) at the time of wounding. Wounds were harvested at post-operative day 7. Frozen sections of the wounds were fixed in cold acetone and stained with a in situ β-gal staining kit. Intense expression of β-gal was observed without any inflammatory response. No significant difference of transfection efficiency was observed between the ischemic and non-ischemic wounds. Thus our data indicates that both AAV and Lentiviral vectors are suitable to use in gene-therapy experiments in both ischemic and non-ischemic cells and tissues in vitro and in vivo.

Notes: Introduction: Hypertrophic scar is a devastating sequel to burns and other tangential skin injuries. It follows deep dermal injuries and does not occur after superficial injuries. Nitric oxide (NO) plays many important roles in wound healing from inflammation to scar remodeling. Studies have shown that expression of nitric oxide synthase and nitric oxide production are decreased in human hypertrophic scar. However little is known about NO involvement in the early stages of hypertrophic scarring, because of the lack of an animal model. It was recently reported that the female red Duroc pig (FRDP) makes thick scar, which is similar to human hypertrophic scar. We hypothesized that NO production in wounds on the female, red Duroc pig is similar to that of human hypertrophic scar and that NO involvement in deep wounds is different from that in superficial wounds. Methods: Superficial (0.015” to 0.030”) and deep (0.045” to 0.060”) wounds were created on the backs of four FRDPs. Biopsies were collected at weeks 1.5, 4, 8 and 21 post wounding including samples of uninjured skin. Nitric oxide levels were measured with the Griess reaction assay and normalized with tissue protein level. Results: Superficial wounds healed with an invisible scar whereas the deep wounds healed with scar resembling mild hypertrophic scar. The thickness of the scars from the deep wounds was significantly greater than uninjured skin and healed superficial wounds (p 〈 0.01). NO levels were increased at 1.5 weeks in deep wounds compared to superficial wounds and uninjured skin (p 〈 0.05). At 8 weeks, NO levels in deep wounds had returned to the level of uninjured tissue and superficial wounds. By 21 weeks, NO levels had decreased significantly when compared to superficial wounds (p 〈 0.01). There were no differences in NO levels between uninjured skin and superficial wounds at any time point (p 〉 0.05).〈inlineGraphic alt="inline image" href="urn:x-wiley:10671927:WRRABSTRACTCY:image_n/WRR_abstractcy_fu1.eps" location="image_n/WRR_abstractcy_fu1.eps" extraInfo="missing"/〉 Conclusions: NO production is similar in late, deep wounds on the female, red Duroc pig to that reported in the literature for human hypertrophic scar further validating this animal model. NO production is quite different after deep wounds as compared to superficial wounds in the FRDP. Early elevation in nitric oxide production might account for excessive inflammation in deep wounds that become thick scars in the FRDP. Nitric oxide regulators and effects at early stages of scar formation should be elucidated further and the FRDP appears to be a useful model.

Notes: Chronic wounds are characterized by a continued inability to heal after an extended period of time (often defined as 3 weeks or longer) and more commonly found in the elderly over areas of compromised blood flow. Collagen is a gene vital to this healing process and Transforming Growth Factor beta 1 (TGFβ1) is an important signaling molecule for collagen induction. We chose to examine the effects of age and ischemia on TGFβ1 regulation using the rat backflap model, which demonstrates negative influences of age and ischemia on the wound healing. Four full-thickness biopsy punches (7 mm) were made centered on the back of aged and young rats. The rostral wound pair was made ischemic by raising a transverse flap (1.8 cm wide × 8 cm length). The caudal wound pair served as a non-ischemic control. Wound contraction was prevented by insertion of a sterilized polyethylene sheet under both the ischemic and nonischemic wounds. RNA was then extracted from wounds at post operation days 3, 7, 10 and 14. The RNA was then reverse transcribed and assayed by Real-Time PCR to examine the relative expression of TGFβ1. TGFβ1 expression in the ischemic wounds of young rats was significantly increased versus nonischemic controls at post-wounding days 3, 7 and 14 (p 〈 0.05, p 〈 0.05, p 〈 0.05, respectively). We also observed a significant increase in TGFβ1 expression in the ischemic of aged animals at day 14 (p = 0.01). The data suggest that there is either a loss of function in TGFβ1’s response to ischemia in the aged animals, or, at least, a significant delay. The results also demonstrate the usefulness of the rat backflap model for the study of age and ischemia on TGFβ1 function in a non-contractile rat wound model.

Notes: Methods to reliably measure tissue oxygenation in situ are currently lacking. We have developed a vertically oriented, dorsal, bipedicle flap model that is easy to perform, reliably reproduces tissue ischemia, eliminates craniocaudal variation, and is amenable to studying therapeutic modalities. The effect of narrowing this flap on tissue oxygenation measured with Licox electrodes has previously been presented. In this study we utilize in situ EPR spectroscopy to demonstrate the oxygen gradient in the flap as a function of flap width and placement of a silicone sheet directly under the flap. The effect of wound healing over a 2 week period is demonstrated.Twenty four, 300 gm male Sprague-Dawley rats underwent creation of the bipedicle flap according to the following groups: 2.5 cm flap with silicone, 2.0 cm flap without silicone, 2.0 cm flap with silicone. Each group of 6 animals was injected with EMS char at 2 cm intervals along the flap and one injection in the control, non-ischemic tissue. A 4th group underwent 2.0 cm flaps with silicone and use of lithium phthalocyanin as the paramagnetic material. Wound measurements and EPR spectroscopy were performed on days 3, 7, 10 and 14. On day 14, after EPR measurements, the animals were sacrificed and their wounds excised. One flap and one control wound were preserved for histologic analysis, the other flap and control wounds were prepared for lactate measurements.EPR spectroscopy demonstrated a gradient of oxygen that was lowest in the center of the flap and greatest at either end. Changes in the oxygen gradient correlated with narrowing and placement of the intervening silicone sheet. This new technology has never been utilized in an animal model of impaired wound healing. Comparison of recently developed paramagnetic materials for optimal tissue oxygen and free radical measurements will be presented.

Notes: Accumulating data strongly suggests that under hypoxic conditions the adaptive physiological response of the cells involves co-operation between oxygen sensing and growth factor signals that cause HIF-1 mediated gene expression. The HIF-1 has been identified as a central and critical molecule in oxygen sensing and possibly a master switch. Of the many cytokines that have been shown to modulate collagen production, TGFβ1 appears to be crucial as it can sustain stimulation of collagen production as well as autoinduction of its own synthesis. Several genes that are known to be hypoxia inducible are also up-regulated by TGFβs, and the promoter of TGFβ3, a member of the family, has a hypoxia-response element (HRE). Recent reports indicate that HIF-1α physically interacts with Smad3 and that the TGFβ and hypoxia signaling pathways synergize at the transcriptional level to regulate gene expression. To determine if this interaction upregulates gene expression through the HRE element, we have co-transfected cultured human dermal fibroblasts with the mammalian expression plasmid for HIF-1α(pCEP1-HIF-1α) with a reporter construct 5HRE-luc, containing a concatemer of five copies of HRE derived from human vascular endothelial growth factor (VEGF), a minimal cytomegalovirus (CMV) promoter and a reporter gene,luciferase.). When treated with 2 ng/ml of TGFβ1 protein, co- transfected hypoxic aged and young human dermal fibroblasts showed significant synergistic upregulation of the reporter gene expression. To further confirm the TGFβ-HIF-1α interaction we have created a TGFβ1 -RNAi construct by subcloning a 19-nucleotide sequence derived from rabbit in a mammalian expression vector (p-SUPER) that directs the synthesis of small interfering RNAs (siRNAs). Hypoxic rabbit cultured dermal fibroblasts co-transfected with TGFβ1-RNAi and 5HRE-luc showed down-regulation of reporter gene expression.As a prerequisite to understanding the biology of physiological and pathological ischemic tissue repair process it is important to delineate the molecular basis of regulation of collagen gene expression in fibroblasts. Each step in the TGFβ signaling cascade is a potential target for highly specific therapy and information about how hypoxic low-oxygen microenvironments affect the TGFβ cascade could be useful to develop novel therapeutic strategies that will involve agonist and antagonist agents that directly interfere with these steps.

Notes: Introduction: The transforming growth factor-beta (TGF-ß) cytokine family regulates cellular proliferation, differentiation, and migration with important function during both development and repair. Moreover, the TGF-ßs are growth inhibitory for keratinocytes, proliferative for fibroblasts, and generally profibrotic during repair. In order to better define the influence of keratinocyte TGF-ß during these processes, we examined the TGF-ß isoform, receptor, and signal messenger Smad expression in fetal and postnatal keratinocytes. Methods: Sprague-Dawley rat keratinocytes were isolated in primary culture from fetal at E15 and E17 (term = E22), newborn, and 6 week old adults. Open fetal rat wounds heal without scar at E17 gestation. Quantitative-polymerase chain reaction was performed for TGF-ß1, -ß2, and -ß3 ligand; TGF-beta receptor1 and -receptor2; Smad3, Smad4, and Smad7 expression. All cells were passage 2 or 3. Results: TGF-ß1 expression did not change appreciably from E15 to adult age in keratinocytes. TGF-ß2 and -ß3 expression increased (8 fold and 2 fold respectively) from E17 to newborn ages. Overall, the expression of TGF-ß1 was 5–8 fold greater compared to -ß2 and -ß3 in both fetal and adult keratinocytes. TGF-ß receptor1 expression increased 2 fold whereas -receptor2 expression showed little change from E17 to adult age. Smad3 expression increased over 50 fold, with Smads 4 and 7 showing a smaller increase from E17 to adult age. Conclusions: The TGF-beta system has differential expression in fetal compared to postnatal keratinocytes, which suggests function during skin differentiation. TGF-ß1 expression is relatively greater than -ß2 and -ß3 in both fetal and adult keratinocytes. However, increases in isoforms -ß2 and -ß3, receptor1 and the Smads occurs at ages associated with scarring, implying increased function of the pro-fibrotic TGF-beta response during ages associated with scarring.

Notes: Background: Physiological differences between dominant and non-dominant arms may play an important role in wound healing of the upper extremities. The Wound Healing Cooperative Group (WHCG) studied this phenomenon in a human forearm model of acute wound healing. Methods: With informed consent, sixteen normal healthy volunteers underwent four 6 mm biopsies on the flexor surface of both forearms: 2 biopsies on the dominant arm, and 2 on the non-dominant arm. The acute wound sites were then treated with i) rhPDGF-BB 0.01% gel topically at various dosing intervals (Q.D. versus Q.O.D.), ii) rhPDGF-BB alone Q.D. × 7 days followed by bacitracin alone, or iii) bacitracin alone. Time-to-complete healing was measured as the primary endpoint. Results: The average time-to-complete healing for the dominant arm was shorter than the time required to heal the biopsies on the non-dominant arm, irregardless of the treatment applied. Conclusion: In the forearm biopsy model of acute wound healing, arm dominance is an independent variable and must considered in study designs. We postulate that this effect may be due to variation in microvascular perfusion. These results provoke further investigation into the physiological mechanisms underlying increased rate of healing in the dominant extremity. Acknowledgements: Funding from The Angiogenesis Foundation.

Notes: Background: Physiological healing in acute wounds generally occurs by granulation from the wound base and by migration of epithelium from the wound edges toward the center of the wound in a uniform, concentric fashion. The Wound Healing Cooperative Group (WHCG) compared this phenomenon in acute wounds treated with standard care versus acute wounds pharmacologically stimulated with growth factor therapy. Methods: With informed consent, 20 normal healthy volunteers underwent four 6 mm biopsies of the flexor surface of both forearms. The biopsy sites were randomly assigned to a control arm (daily bacitracin) or to one of three treatment arms: i) rhPDGF-BB 0.01% gel Q.D., ii) rhPDGF-BB Q.O.D., iii) daily rhPDGF-BB (Q.D. × 7 days followed by bacitracin alone daily). Wound morphology was carefully examined and photographed daily until complete healing was achieved. Results: There were distinct differences in the morphological pattern of healing seen between control wounds and growth factor-stimulated wounds. Acute wounds treated with bacitracin tended to heal in circumferential fashion, as predicted. Growth factor-stimulated wounds, by contrast, exhibited accentuated angiogenesis (granulation), with non-uniform epithelial islands streaming into the wound. Conclusion: This pilot study suggests that rhPDGF-BB influences acute wound healing by promoting accelerated granulation and epithelialization. Accelerated healing is manifest by different healing morphologies. The biological basis for these differences requires further histological and molecular analyses. Acknowledgements: Funding from The Angiogenesis Foundation.

Notes: The inability to experiment in humans creates a need to develop culture systems that mimic human tissues/organs. Skin is arguably the simplest tissue hence provides an excellent prototype for tissue engineering. Moreover, skin is the body’s first line of defense. Currently available skin replacements have a number of drawbacks. Therefore, there is a need for skin replacements that: (a) are prepared with pertinent primary human cells but yet can be ready “off” the shelf; (b) can be prepared rapidly; (c) contain stable structures, in particular microvessels that can rapidly connect with the patient’s vasculature, thereby establishing circulation in the “graft” and increasing the chances of survival; (d) can be tailored for specific wound impairments (e) are long lasting. We have developed a generation human “skin” that can fulfill these requirements and can potentially be used as a “living bandage”. We start with three primary human cell types and a collagen matrix that self-assemble into a connective tissue containing a network of mature microvessels, is covered with a stratified epidermis, expresses biochemical markers, matrix molecules, and cytokines characteristic of normal human skin, and matures in 10–15days. Moreover, two additional cell types, pericytes and monocytes, differentiate in situ adjacent to and within microvessels, respectively. The epidermis expresses keratins typical of mature skin and not characteristically produced in response to injury such as keratins 6, 16, 17. This tissue potentially can be developed into a skin replacement for patients with impaired healing. In addition, it responds normally to biological stimuli, providing a powerful vehicle to investigate mechanisms of skin development and regeneration, understand pathological processes, and test drugs and treatments for skin diseases.

Notes: Delayed wound healing is a common complication of diabetes resulting in significant clinical morbidity. The diabetic wound exhibits impaired cellular infiltration and consequently inadequate granulation tissue formation. Additionally, altered patterns of apoptosis. In this study, we have evaluated the differential gene expression pattern in transgenic diabetic female mice, 5–6 weeks old, in a full thickness cutaneous punch wound model. We assessed the role of matrix metalloproteinases (MMPs) and differential gene expression pattern at 4th, 7th and 11th day post wounding. Supernatants obtained from diabetic wound tissue homogenates were subjected to zymogram analysis. The data showed that MMPs were expressed at higher level by 4th day post wounding, whereas expression of MMPs were down regulated towards the 11th day post wounding suggesting their role during early phase of wound healing.The pathway specific gene array data demonstrated differential regulation of several growth factors, transcription factors and other related genes such as fibroblast growth factors and their receptors, ID3 and restin respectively. The cytokine/extracellular matrix protein osteopontin (OPN), an important component of cellular immunity and inflammation also showed higher expression after 4 days post wounding. The expression of OPN remained at higher level after 11 days post wounding in diabetic mice, whereas the expression were down regulated to basal level in normal wounded animal suggesting that the expression of OPN was concomitant with the extent of healing. Other adhesion molecules such as integrin αV and PECAM-1 were also differentially regulated. Though a single gene may not be solely responsible for any defect or impairment in healing as it is a very tightly controlled and regulated process, however, a detailed study of these gene(s) may shed some light to the delayed healing in diabetic mice. Acknowledgements: (These studies are supported by NIH Grant G174KT.)

Notes: Venous leg ulcers derived from tissue destruction is the consequence of a chronic inflammatory process that produces pain and physical disability, diminishing quality of life in patients. In this work, Lassar ointment and lyophilized collagen-polyvinylpyrrolidone were administered separated each on one half in the same ulcer to 9 patients at the beginning and every 4 days. On day 16, all patients were auto-grafted with partial thickness skin. Granulation tissue and graft integration were assessed clinically during 3 months. Inflammatory infiltrate, type I and III collagens, elastic fibers, alkaline phosphatase as well as blood vessels were evaluated histologically or histochemically in biopsies taken at the beginning and 16 days after the local treatment.Clinically and morphologically, both treatments demonstrated appropriate granulation tissue promotion and optimal graft integration since the beginning. Nevertheless, in Lassar ointment treated group regionalization of alkaline phosphatase activity was observed, as well as the presence of granuloma in 2 of the 9 patients. In conclusion, Lassar ointment or lyophilized collagen-polyvinylpyrrolidone are two different promoters of granulation tissue in venous leg ulcers, however Lassar ointment has the capability to produce granuloma and an exacerbated immune response; in consequence, ulcer recidivism could be present, may be due to mineral deposits in the wound.

Notes: Celosia argentea is used in traditional medicine for sores, ulcers, and skin eruptions. Therefore, we investigated the healing efficiency of an ointment formulated on a alcohol extract of Celosia argentea leaves (CA) in a rat burn wound model. Wound closure occurred earlier in the treated rats (15 days versus 30 in the untreated group; P 〈 0.05). Collagen and hexsoamine content of the granulation tissue increased at a faster rate in the treated wounds. To probe the cell biologic basis of this effect, we found that this extract promoted cell motility and proliferation of primary dermal fibroblasts but did not alter these responses in primary keratinocytes. In short, we demonstrate a salutary action of the Celosia argentea extract on wound healing and suggest that this may be due to mitogenic and motogenic promotion of dermal fibroblasts.

Notes: The lack of understanding of molecular pathogenesis of impaired healing in chronic ulcers leads to a serious health issue that contributes to excessive limb amputations and mortality. Using biopsies from patients with chronic wounds, skin organ culture and primary keratinocytes in culture we identified that β-catenin and its downstream target, c-myc, play important role in development of chronic wounds. In contrast to normal epidermis, we observed significant nuclearization of β-catenin and elevated c-myc expression at the non-healing wound edge of patients with chronic ulcers. In vitro studies indicated that activation and stabilization of nuclear β-catenin inhibits wound healing and keratinocyte migration by: blocking EGF response and inducing c-myc. Using Affymetrix large scale microarrays we found that β-catenin downstream target, c-myc, is induced in skin by an inhibitor of wound healing (glucocorticoids) and repressed in the initial phase of normal wound healing, 4 to 48 hr, whereas it becomes de-repressed at 96 hr post wounding. Therefore, the activation of β-catenin/c-myc pathway(s) contributes to impaired healing by inhibiting of keratinocyte migration and altering keratinocyte differentiation. The presence of activated β-catenin and c-myc in the epidermis of chronic wounds may serve as molecular markers of impaired healing and future targets for therapeutic intervention. While β-catenin signaling has been implicated in epithelial development and oncogenesis its role in wound healing has never been postulated. This further illustrates the importance of “tissue context” specificity, because β-catenin in the context of malignant tissue promotes invasion whereas in the context of a wound environment does the opposite.

Notes: Keloids are scars that overgrow the original boundaries of the injury, involving aberrant functioning of the fibroblasts. We have found that during normal wound healing the ELR-negative CXC chemokines IP-9 (CXCL11) and IP-10 (CXCL10) act to limit fibroblast immigration. Our initial goal was to determine if keloid fibroblasts failed to respond to this ‘stop’ signal. Immunohistochemical and immunocytochemical studies on keloid fibroblasts show that these lesions express excessive amounts of IP-9 and CXCR3 receptor. The protein levels of CXCR3 receptor was also not dissimilar between normal and keloid fibroblasts. Though, the expression of the protein and the receptor seems to be normal, the fibroblasts derived from these abnormal lesions did not respond to IP-9 during EGF induced cell migration. We have extended these findings to determine whether other genes are differentially expressed by keloid fibroblasts. The immediate first gene that interest us was the mRNA levels of CXCR3 receptor (GPR9) wherein, we did not find any significant difference between normal and keloid fibroblasts. Further, we also found interestingly, that genes related to tumor progression are up regulated in keloid fibroblasts than the normal fibroblasts. The marker genes like calgranulin, BCL-2 associated anthogen 4 (BAG4), and dual specificity phosphatase 1 (DUSP1). Certain transcription factors like GATA-6 and SpiB are also increased. Apart, the classical growth factor and growth factor receptor related genes which includes, bone morphogenetic protein BMP-1, heparin binding EGF like growth factor, EGF like repeats discoidin1, IGFBP-5 and 3, EphB1&B2, tumor necrosis factor alpha induced protein, protein tyrosine phosphatase receptor, serine threonine kinase 6 along with chondroitin sulfate proteoglycan brevican and versican and vitronectin are also up regulated. The newl identified genes will open new avenues in targeting keloid lesions by understanding the basic signaling mechanism of these specific proteins.This work was supported by grants from NIH/NIGMS and PPRTP.

Notes: Peptides of the innate immune system play a vital role in the protection and repair of almost all biological systems. Such peptides have been implicated in a range of activities associated with prevention of disease and modulation of innate immunity. HB107 is a derivative of one such peptide, Cecropin B, that has demonstrated efficacy in enhancing wound healing in both burn and incision animal models. HB107 has been evaluated for efficacy in a mouse incision model and for safety and efficacy in a pig burn wound model. Topical application of the peptide gives RE50(time needed for 50% re-epithelialization) values of 10.28 days for 500 ug/mL and 12.72 days for 100 ug/mL compared to control 16.45 days. Additionally the peptide was well tolerated in terms of safety both topically and in an IV acute toxicity mouse model with no adverse effects observed. HB107 not only demonstrated efficacy and safety, but due to being a relatively short synthetic peptide, costs significantly less to manufacture than the current approved therapies.

Notes: Intact skin represents the first line of defense against microbial pathogens. Effectors of innate immunity such as antimicrobial peptides are important when this barrier is breached due to infection or injury. Antimicrobial peptides can kill microbes directly, however several investigations have suggested they can also modify host cell behaviors to promote wound repair. It is unclear whether the ability of these molecules to positively influence wound repair is dependent on their antimicrobial function or on their ability to influence the host. To investigate this, the microbial killing capacity of specific antimicrobial peptide fragments was determined and compared with their ability to affect wound repair in a murine model of aseptic full-thickness excisional injury. HB-107, a peptide fragment derived from Cecropin B, lacks antimicrobial activity yet showed up to 64% improvement in wound closure at day13 when compared to either scrambled peptide or vehicle controls. This effect was comparable to that seen after treatment with currently accepted therapy. Histological evaluation of wounds treated with HB-107 displayed keratinocyte hyperplasia and increased leukocyte infiltration compared to controls. To explore the mechanism for these findings, we tested the ability of HB-107 to stimulate IL-8 secretion. HB-107 peptide was able to stimulate IL-8 release from cultured dermal microvascular endothelial cells when compared to scrambled peptide control or other peptides derived from Cecropin B. Taken together, this data confirms that antimicrobial peptides can function as important effectors of wound repair independent of antimicrobial function.

Notes: Calreticulin (CRT) is a major classic Ca-binding chaperone protein of the endoplasmic reticulum. Recently, CRT has been recognized to have widespread extracellular effects as well. In the current study we show that CRT increases both epithelial migration and granulation tissue formation in models of porcine and murine wound repair. Partial thickness wounds were created on the paravertebral area of pigs (n = 4) and 0.1% and 0.5% CRT, and PDGF (positive control) applied for 4 consecutive days. In wounds harvested at 5 days, CRT induced a 28 and 22% greater extent of reepithelialization than PDGF and Tris/Ca buffer control, respectively (% healed = 56/CRT; 40.5/PDGF; 44/control). In addition, CRT stimulated earlier granulation tissue formation in a dose-dependent manner (cumulative dermal depth, microns: 1615/CRT; 1250/PDGF; 1325/control). A similar granulation tissue inducing effect of CRT was also observed in a steroid-impaired pig model. As a diabetic model of wound repair, two 5 mm circular full-thickness wounds were created on the dorsum of db/db mice; the wounds were splinted open with silicone rings and covered with occlusive dressing (n = 24). After 5 days of treatment with 0.1, 0.5, and 5% CRT, a dose-dependent 8-, 4.5-, and 2-fold increase in granulation tissue formation was observed (p 〈 0.05). However, there was no apparent effect on wound closure. Tissue sections showed a highly cellular dermis in the CRT treated wounds. In addition, CRT (50 pg/ml) stimulated wound closure in a scratch plate assay using fibroblasts by 45%, in 48 hrs, compared to 2% for the control. Therefore, CRT may be a novel agent for wound healing by acting as a chemoattractant for cells involved in wound remodeling and in epithelial migration.Suppported by Calretex, LLC. NJ, USA (LIG) and the Alumni Fund, Alumni Association Downstate College of Medicine (MJC).

Notes: Bone marrow cells have been used to potentiate wound healing in chronic wounds (Badiavas and Falanga, 2003). We postulate that the performance of these autografted cells may be enhanced by seeding them into scaffolding with normal skin architecture; indeed that the differentiation of the precursor cells may be determined by the juxtaposition of intact extracellular matrix. This might in turn lead to a prefabricated skin substitute capable of recapitulating fully functional skin.We are seeding murine gfp+ bone marrow cells, containing mixed mesenchymal and hematopoietic precursor cells into Alloderm®, a decellularized preserved skin graft. This seeding is potentiated by meshing the skin and gentle centrifugation of fresh marrow into the dermal aspect of the allograft. Other samples were merely co-cultured with the cells in Dexter’s media. Duplicate samples were fixed and stained with DAPI at 1, 3, 7, and 14 days. Viewed under fluorescence, gfp+ cells are bright green.Comparing these images with DAPI-staining allows viable cells to be identified which are gfp+. H&E micrographs were used to ascertain cellular morphology. Results: Scant cells were observed in the allografts at the early time points. The cells were pleiomorphic with large nuclei, resembling hematopoietic precursors(〈link href="#fu1"〉Fig 1). Later, at days 7 and 14, more abundant cells were apparent, including some in the interior of the dermis. This signifies either migration or seeding via centrifugation. The morphology of these cells is significantly different; the spindle shape suggests a fibroblast (〈link href="#fu2"〉Fig 2). In addition, the population of gfp+ cells has markedly expanded by day 14, attaining near confluence in some sections (〈link href="#fu3"〉Fig 3). No cells were seen in controls (〈link href="#fu4"〉Fig 4). Conclusion: True skin replacement will only be possible when the panoply of skin appendages can be recapitulated. The experiments presented here are evidence that pluripotential precursor cells can be seeded into dermal matrices providing an optimal environment for regeneration.〈figure xml:id="fu1"〉1〈mediaResource alt="image" href="urn:x-wiley:10671927:WRRABSTRACTEP:image_n/WRR_abstractep_fu1.eps"/〉〈figure xml:id="fu2"〉2〈mediaResource alt="image" href="urn:x-wiley:10671927:WRRABSTRACTEP:image_n/WRR_abstractep_fu2.eps"/〉〈figure xml:id="fu3"〉3〈mediaResource alt="image" href="urn:x-wiley:10671927:WRRABSTRACTEP:image_n/WRR_abstractep_fu3.eps"/〉〈figure xml:id="fu4"〉4〈mediaResource alt="image" href="urn:x-wiley:10671927:WRRABSTRACTEP:image_n/WRR_abstractep_fu4.eps"/〉

Notes: Maththaan thailam, the Datura leaf juice is traditionally used for healing of wounds and related ailments in India. However, the scientific validity of the thailam has not been explored in the literatures. In the present study, the efficacy of maththaan thailam on healing of three types of wounds namely Open, Burn and Diabetic wound in animal models has been studied. Healing pattern and biochemical characterization of granulation tissue were assessed over the healing periods. Studies were more concentrated on to evaluate the change in the matrix metallo proteinase profile in the three different types of wound with respect to the applied thailam. Further supports were also expected from the antioxidant levels and histopathological examination of granulation tissue. It has been observed that among the three types of wounds, delayed healing was observed with diabetic wound. Complete healing of 2 × 2 cm2 wound size was achieved only after 22 days, whereas burn and open wound took only 16 and 9 days for complete closure. Further, MMP’s profile also showed a variation in the expression of MMP2 and MMP9 with respect to healing property. Biochemical and histochemical results and the antioxidant levels are highly correlated with the healing property irrespective of the wounds

Notes: Introduction: We hypothesize that interleukin-6 receptor (IL-6r) blockade with IL-6r antibody will increase matrix metalloproteinase-3 (MMP-3) production in hypertrophic scar fibroblasts.To test this hypothesis, MMP-3 protein levels were measured in fibroblasts cultured from normal skin and hypertrophic scar with and without IL-6 receptor blockade. Methods: Primary cell cultures of normal skin (NSF) and hypertrophic scar fibroblasts (HTS) were obtained from the same patient (n = 5) and seeded into 6 well plates (40,000 cells/cm2) in complete media. Cells were treated with and without IL-6 receptor antibody and IgG (10 ng/ml) for 6 hours. Protein concentrations of MMP-3 are determined in equal volume of supernatants by ELISA.Statistical analysis was by unpaired t-test(*P 〈 0.05). Results: Blocking IL-6 receptors with its antibody showed significant increase in MMP-3 protein concentration in HTS compared to NSF.〈table frame="bottom"〉〈tgroup cols="4" align="left"〉〈colspec colnum="1" colname="col1" align="left"/〉〈colspec colnum="2" colname="col2" align="left"/〉〈colspec colnum="3" colname="col3" align="left"/〉〈colspec colnum="4" colname="col4" align="left"/〉〈thead valign="bottom"〉〈row rowsep="1"〉MMP-3(ng/ml)MMP-3(ng/ml)〈tbody valign="top"〉NSF1.6 ± 0.13HTS0.7 ± 0.37NSF + IL-6r0.3 ± 0.15HTS + IL-6r2.3 ± 0.5*NSF + IgG0.26 ± 0.16HTS + IgG0.75 ± 0.14 Conclusion: IL-6 receptor mediated signaling is involved in the pathogenesis of hypertrophic scar formation and its blockade may reduce hypertrophic scar formation.

Notes: Background: Apligraf, a bioengineered living skin construct, has been shown to accelerate the healing of chronic venous leg and diabetic foot ulcerations. However, to date the mechanism of action of Apligraf in the wound healing process is not well understood. Objective: The primary objective of this study was to determine the levels of expression of selected wound healing related genes in venous leg ulcers treated with Apligraf in comparison to ulcers treated with standard multi-layer compression therapy alone. Gene chip technology was employed. Methods: Three patients were randomized into the Apligraf or standard treatment arms. A baseline 6 mm punch biopsy was obtained prior to the initial application of Apligraf or multi-layer compression therapy. A second biopsy was obtained depending on the randomly chosen biopsy schedule (weeks 1, 2 or 4 following Apligraf application or initiation of compression). The biopsy specimens were snap frozen and later analyzed using microarray gene chip technology. Results: The patients treated with Apligraf demonstrated an up-regulation of the genes thought to be important in the wound healing process. Conclusion: The results from this pilot study suggest that Apligraf may function by an up-regulation of the genes involved in wound healing as opposed to compression therapy which works primarily in a mechanical fashion. Based on these results further study is needed.

Notes: To explore the effects of bone marrow mesenchymal stem cells (MSCs) on the quality healing of porcine skin wounds after burn injury so as to provide a new method for clinical skin repair in the future. Seventy-two deep-partial thickness burn wounds were produced on the back of 6 minipigs and randomly divided into 6 groups: saline control, MSCs treatment, MSCs plus bFGF treatment, MSCs plus EGF treatment, bFGF treatment or EGF treatment only. MSCs were isolated from porcine marrow and cultured in vitro. After labeling with BrdU, MSCs were autografted onto the skin wounds. At 7, 14, 21 and 42 days after injury, the area of the wounds were measured and the histological examination was performed to evaluate the velocity and quality of wound healing. At 1, 2 and 4 weeks after transplantation, immunohistochemical examinations were carried out to detect the positive staining of BrdU, cytokeratin and S-100 to evaluate the wound healing quality. The area of wounds was decreased at day 7 and most of these wounds were healed on day 21 after injury. There was no significant difference on the contraction rate among six groups. Histological examination demonstrated that the number of vessel and the expression density of S-100 in MSCs plus bFGF treatment wounds were significantly enhanced than that in other groups. MSCs autografting may benefit to enhance the wound healing quality in porcine skin, which may open a new way to reach a “perfect repair” after skin injury.

Notes: The onset of infection can lead rapidly to sepsis, septic shock, and eventually death. Considering the high costs of hospitalization and the added trauma and discomfort to the patient, improved methods for infection control are needed. Ozone offers a specific solution to the problem of effective management of microbial wound contamination. Despite the advantages, several technical barriers have prevented ozone-based disinfection treatments from becoming more widely investigated. Negative press from years of unsubstantiated medical successes has made the medical community wary of even legitimate ozone technologies. Because there has been no research into using ozone in infection prevention, much of the hardware has not been developed to the stage where the technology can be used in clinical trials. Lynntech Inc., over the past 5 years, has been working to develop a pre-clinical ozone-based wound management system. This new hardware uses an electrochemical ozone generator that can deliver a predetermined quantity of ozone gas to a specific wound site. Electrochemically generated ozone has been shown to be effective against a range of gram negative and gram positive bacteria and our studies with various mimetic wound systems have shown that with controlled delivery, ozone can be utilized as either a disinfectant or as a biostat. The device is well suited for hospital use as it operates of low voltage power supplies and unlike other ozone generation technologies will not interfere with other electronic equipment while it is in operation. This paper outlines both the advantages and limitations of using an ozone based system as a wound management tool and looks to the future research that needs to be performed to substantiated the initial research findings.

Notes: Bone marrow is an important supply for many progenitor cells, among them endothelial precursor cells. They have been already considered as potential therapeutic angiogenesis for ischemic hindlimb. In this work we performed an unilateral chronic ischemic hindlimb model in 20 dogs, we were dissected and ligated the middle sacra artery and external right iliac artery preserving the internal iliac artery, after one week we removed de femoral artery performing a proximal and distal ligation and inserted into the gracilis muscle 3 Silastic tubes (0.8 mm diameter) in a middle circle form in order to created fibrocollagenous tunnels. Fifteen days later, we obtained bone marrow (30 mL) and mononuclear cells were separated; at the same time, all tubes were removed and bone marrow cells were transplanted into the fibrocollagenous tunnels, only in the third and fourth groups. During the 8 at 12 days we administered subcutaneously saline solution to the first and third groups and G-CSF to the second and fourth groups. Finally, after 30 days we performed contrasted angiographies from both limbs and was calculated mean angiographic score (MAS) from the number of vascular intersections, also was taken a biopsy from the central portion of right gracilis muscle for vascular assessment, as well as for vascular proliferating cells by immunohistochemistry.Results demonstrated that white bone marrow cell transplantation enrichment by G-CSF administration stimulates angiogenesis in ischemic hindlimb, twice than controls, and it is better than white bone marrow cell transplantation alone. White bone marrow cell is a potential therapy for ischemic hindlimb, because stem cells are growth factor provider, as well as an important source for endothelial precursors capable to form vessels de novo, mainly when this stem cells are deposited in an appropriate extracellular matrix, such as the fibrocollagenous tunnels in this model.

Notes: The inability to experiment in humans creates a great need to develop culture systems that mimic human tissues/organs. Skin is arguably the simplest human tissue and therefore provides an excellent prototype for tissue engineering. Moreover, skin is the body’s first line of defense.Currently available skin replacements have been classified into four categories: (i) those that are composed completely of epidermal cells; (ii) those consisting of dermal components derived from processing of cadaver skin or from collagen and other matrix molecules; (iii) those containing both dermal and epidermal components and (iv) those that contain dermal, epidermal and vascular components. All of these substitutes have drawbacks. Therefore, there is a need for skin replacements that: (a) are prepared with pertinent primary human cells but yet can be ready “off” the shelf; (b) can be prepared rapidly; (c) contain stable structures in particular microvessels that can rapidly connect with the patient’s vasculature in this manner establish circulation in the “graft” increasing the chances of survival; (d) can be tailored for specific wound impairments (e) are long lasting. We have developed a new generation human “skin” that can fulfil these requirements and can potentially be used as a “living bandage”. We start with three primary human cell types and a collagen matrix that self-assemble into a connective tissue containing a network of mature microvessels, is covered with a stratified epidermis, expresses biochemical markers, matrix molecules, and cytokines characteristic of normal human skin and matures in 10–15 days. Moreover, two additional cell types, pericytes and monocytes, differentiate in situ adjacent to and within microvessels, respectively providing stability to the micriovessels and the epidermis expresses keratins that are typical of mature skin and not those characteristically produced in response to injury such as keratins 6, 16, 17. This tissue can potentially be developed into a skin replacement for patients with impaired healing. In addition, this tissue responds normally to bological stimuli, providing a powerful vehicle to investigate mechanisms of skin development and regeneration, understand pathological processes, and test drugs and treatments for skin diseases

Notes: Previous studies have shown that angiotensin peptides, NorLeu3-A(1–7) in particular, accelerate dermal healing and reduce scar formation. In this report, the effect of this peptide on scar formation is more fully delineated. The effect of surgical day, time after injury and observer on the clinical appearance of the incision was determined. Clinical observations of incision site included inflammation, dehiscence of the injury and appearance of scar were conducted by two blinded observers (two observations per time point) twice weekly. The scores correlated between observers and for the same observer within an observation day. Dehiscence of the incision occurred in 35% to 40% of incisions early (days 4 and 7) after injury. Administration of NorLeu3-A(1–7) at the time of injury reduced the incidence of dehiscence at day 7 to approximately 20%. Further, the length of the wound opening was significantly reduced in the peptide-treated incisions at day 7. Starting on day 14 after injury, scar formation was evaluated. Up to 80–90% of control animals had observable scars starting on day 14. Thereafter, the scar remodeled with fewer incisions having visible scar on day 28. With administration of NorLeu3-A(1–7), significantly fewer incisions had observable scars starting on day 14 and throughout the study. As few as 20% of the incisions had observable scars on day 28. The histological appearance of the healing wound was also evaluated at weekly intervals starting on day 7 and continuing until day 42. At day 7, the maximal number of fibroblasts at the wound site was observed. Thereafter, the number gradually reduced, plateauing at day 28. The administration of peptide had no effect on fibroblast number at the incision site. A similar pattern was observed in the thickness of the epidermis with the resolution of te hyperplastic phase at day 21. Administration of the peptide significantly increased epidermal height at day 7. Blood vessel formation peaked on day 21 and 28 in control wounds and was further enhanced by peptide administration during the neovascularization phase. After day 28, blood vessel number was comparable between control and treated incisions. Collagen deposition and remodeling were are increased by the administration of NorLeu3-A(1–7) at the time of injury. This paper describes the kinetics of scar formation in the rat by clinical and histological observation and further described the beneficial effect of NorLeu3-A(1–7) on scar formation.

Notes: Normal wound healing is a carefully controlled balance of new tissue formation and destructive processes necessary to remove damaged tissue. Within this complex environment there are many points of regulation, which control the biological processes necessary to achieve wound repair. An alteration in any of these processes can result in an imbalance of the biochemical components, which ultimately results in delayed wound closure and the formation of a chronic wound. Therefore, we propose that an effective therapeutic approach would modify this hostile environment and redress this imbalance.In this study we have evaluated the effect of PROMOGRAN in vivo, in patients with venous leg ulcers. Wound fluid samples were collected and analyzed from a number of patients prior to and during treatment with PROMOGRAN.Our results have shown that wounds, which respond to PROMOGRAN treatment, have also shown a decrease in protease activity in the corresponding wound fluid samples. Whilst it is impossible to determine whether this reduction in protease levels is responsible for healing or merely symptomatic of other changes occurring within the wound, we have shown that a decrease in proteolytic activity is concomitant with healing.This study provides clinical evidence that PROMOGRAN can rebalance the chronic wound environment in situ and thereby promotes wound repair.

Notes: Fibrin Sealant products such as TisseelR have been used over the last 25 years to reach hemostasis during surgery or to seal tissues. Moreover, there were many studies on the use of fibrin sealant in cell and bioactive substance delivery. Our In Vitro and In Vivo studies, over the last 2 years, have focused on the use of fibrin sealant to deliver human fibroblasts or keratinocytes to overcome the healing deficiency in chronic wounds. We have shown that some fibrin formulations supported a high fibroblast proliferation and other fibrin formulations supported a high proliferation of human keratinocytes. In this study, we examined the use of fibrin sealant in the co-delivery of both human – derived keratinocytes and fibroblasts. The study report examined the cell proliferation of these two cell types in various formulations of fibrin sealant. Fibroblasts and keratinocytes were mixed with various dilutions of the Sealer Protein solution and added to culture plates before adding the Thrombin solution to form fibrin clots containing both cell types. We found that a low to medium sealer protein concentration (1–34 mG/mL) and a very low thrombin concentration (1 U/mL) in the final fibrin clots provided for an optimal cell proliferation for both cell types within these fibrin clots. This profile of proliferation was different from that seen when keratinocytes alone or fibroblasts alone were incorporated in the fibrin clots. Morphologically, it was difficult to determine the cell type from examining cell morphology, thus, it was difficult to determine the cell distribution. In conclusion, we found that various modified formulations of fibrin sealant may be chosen when co- delivering fibroblasts and keratinocytes. Moreover, there seems to be a positive feedback between keratinocytes and fibroblasts when they were co – introduced in the fibrin clots. This feedback could be carried by growth factors. Future studies will determine his signaling process.

Notes: Background: More than three quarters of patients with a connective tissue disorder will develop secondary Raynauld’s disease. This more severe form of the Raynauld’s can lead to digital ischemia, tissue loss and may even result in the need for amputation of one or more digits. Cilostazol is a phosphodiesterase inhibitor currently approved for use in vascular disease of the lower extremities. Case History: In August of 2002 a 62 year old female pianist with scleroderma presented to the wound clinic with a two year history of intractable digital pain secondary to ischemic ulcerations of the index, long and ring fingers of the right hand. She suffered from secondary Raynauld’s disease. Prior unsuccessful treatments included warming the hands, calcium channel blockade, anticoagulation, topical xylocaine and narcotic analgesics. In August of 2002 cilostazol was started at 100mg BID. Wound care consisted of serial debridement, moist wound healing and topical rhPDGF. Results: Four weeks into treatment the patient’s narcotic requirement decreased substantially and the fingers had a pink appearance. The ring finger healed after eight weeks. At three months the patient discontinued narcotic pain medication. Nine months into treatment the index finger had healed and she resumed playing the piano. The long finger finally healed one year after she initially presented to the wound clinic. Conclusion: Cilostazol may be an important adjunct in the treatment of secondary Raynauld’s disease particularly when digital ischemia has developed. Acknowledgements: Funding from Otsuka Pharmaceuticals.

Notes: Identifying molecular loci of impaired cutaneous healing in diabetes with an eye towards developing targeted therapy to ameliorate dysrepair continues to evolve as a promising area of study. By using an excisional wound model produced on the dorsum of female diabetic C57BL/KsJ db + /db+ mice as well as their normal (WT) & heterozygous (HZ) littermates, we studied the effects of peri-wound intradermal injection of adeno-associated viral vector (AAV) expressing the 165-amino acid isoform of human vascular endothelial growth factor (VEGF) on the following: kinetics of re-epithelialization, neoangiogenesis and granulation tissue formation, matrix remodelling, collagen deposition, and maturation. One sq. in. full thickness excisional wound was created in the mid-upper back, rendering half of the wound as either right or left paravertebral. Animals were randomized to receive 1 of 3 treatments via intradermal injection: 1)VEGF (AAV) vector; 2)Adnull vector; 3)PBS. Postoperatively, wounds were examined & photographed on Days 3, 7, 10, 14, 21 & 28. Also, tissue was harvested for histology & immunohistochemistry (PECAM), and snap frozen for protein & RNA analysis. A scoring system was used to grade re-epithelialization, granulation tissue thickness, matrix density, inflammation, vascular density, epithelial maturity. AAV-VEGF exerted minimal effect on repair in WT and HZ mice. However, pronounced neovascularization, thickened granulation tissue & increased matrix deposition was noted after VEGF treatment in the db/db mice compared to those that received PBS or adnull vector at all timepoints. While the induction of angiogenesis in VEGF treated db/db mice lagged behind the unimpaired mice by 5–7 days, a global improvement in wound healng was observed.R Crystal, Dir Inst Genetic Medicine, Weill Med College-Cornell Univ

Notes: Macrophages serve as the coordinators of the wound healing process. Since 1998 following the Israeli Ministry of health authorization, macrophage suspensions have been used for the treatment of ulcers, in more than 800 elderly and paraplegic patients suffering from decubital chronic ulcers.As previously published, a significant number of genes showed increased levels of expression in hypo-osmotic shock activated cells, using DNA microarrays technique. The majority of these genes are considered to be directly involved in the macrophage function and in the wound healing process.Macrophge suspensions are prepared from a whole blood unit of healthy, young volunteer blood donors in a closed, sterile system, as previously described. The activated cells are applied to the wounds either by local injection or by direct deposition to the wound.Between January 2000 and October 2003, 112 patients with postoperative sternal wound infection were treated with macrophage suspension. Full closure of the wounds was achieved in 104 (93%) of the patients.〈table frame="topbot"〉〈tgroup cols="2" align="left"〉〈colspec colnum="1" colname="col1" align="left"/〉〈colspec colnum="2" colname="col2" align="left"/〉〈tbody valign="top"〉Original wound surface area8–175 cm2 (mean 93)Days until treatment6–180 (mean 47)Days until 50% closure6–60 (mean 21)Days until full closure10–138 (mean 49) No side effects were noted.The use of macrophage suspension is a safe and effective therapeutic strategy that reduces risk of complications and morbidity and improves the quality of life for long -suffering patients. Length and cost of hospital stay may be reduced, as the treatment requires no hospitalization.

Notes: Matrix metalloproteinase-9 (MMP-9) transiently expresses in acute wound. In non-healed wounds, MMP-9 together with other proteinases persistently elevate, which may lead excessive ECM degradation and failure of wound closure. To understand the molecular regulation of MMP-9 we investigated the signal transduction for TNF-alpha mediated induction of MMP-9 by dermal fibroblasts. TNF-alpha initiates three major signal pathways including NF-11B, JUN N-terminal kinase (JNK), and p38 MAPK. On the other hand, Rho-GTPase plays an important role in a variety of cellular functions including cell morphogenesis, motility, survival, angiogenesis, and mitosis. It remains unknown if the “cross talk” of these signals having a role in regulation of matrix metalloproteinases (MMPs). In this study we found that over expression of the p21-activated kinase (PAK) specifically attenuates TNF-alpha mediated induction of MMP-9. However, TNF-alpha mediated induction of MMP-3 and proMMP-2 activation was intact. NF-κB signal is regarded as a common pathway for many MMPs. Indeed, PAK did not affect TNF-alpha mediated degradation of Ikappa B, suggesting additional signal is targeted by PAK. In contrast, MMP-3 but not MMP-9 expression is specifically blocked by p38 MAK. Thus TNF-alpha induced expression of multiple MMPs in wound healing may utilize different intracellular signal pathways.

Notes: Fetal dermal wound healing is characterized by a minimal inflammatory response and absence of scar. In order to determine the factors contributing to the minimal inflammatory response in the fetus, the role of P-selectin in the interaction and transmigration of leukocytes through vascular endothelium was investigated.
Methods: Primary endothelial monolayers were established from adult porcine central veins and from umbilical veins of mid-gestation porcine fetuses. The ability of the endothelial cells to capture and enable transmigration of adult leukocytes was studied under flow conditions of 4 dynes/sec, with and without prior stimulation of the endothelial cells with TNF-α or IL-1β. In addition, P-selectin mRNA expression was determined by quantitative real time PCR. All data were analyzed by ANOVA. 
Results: In response to IL-1β 10 ng/ml stimulation, adult endothelial cells manifested a 10 fold increase in P-selectin mRNA expression while fetal endothelial cells mounted only a 3.5 fold increase. 100 ng/ml was required to mount a 10 fold increase in fetal P-selectin expression while no further increase in adult P-selectin expression was noted at this dose. Leukocyte capture, demonstrated by rolling of neutrophils over the endothelial surface, was more efficient on adult monolayers compared to the fetus. This is inversely related to the rolling velocity which was significantly slower in the adult (5.3 ± 0.6 vs 12.4 ± 1.0 μm/mm2). Ultimately, the number of neutrophils transmigrating across adult endothelial monolayers under flow conditions was significantly higher compared to the fetal monolayer (199 ± 18 vs 72 ± 9 cells/mm2).
Conclusion: Lower P-selectin expression on fetal endothelial cells may account, in part, for the minimal inflammatory response noted following fetal dermal injury.

Notes: Although collagen wound dressings have been used clinically for many years, wound matrix materials comprising the full complement of extracellular matrix molecules have not. Oasis® Wound Matrix (Oasis), a complex, growth factor-containing matrix derived from the intestinal submucosa of pigs, has shown promise as a treatment to manage full-thickness, hard to heal wounds. A multi-center, prospective, randomized clinical trial was conducted to compare the effectiveness of Oasis to a standard of care (SOC) regimen consisting of compression therapy, weekly dressing changes and debridement (as needed) for the treatment of venous leg ulcers. Incidence of healing within 12 weeks, as defined by full epithelialization without drainage, was the primary outcome measure. Significance was determined by the proportion of ulcers healed using Fisher’s Exact Test. This interim data included 84 evaluable patients (45 Oasis, 39 SOC). Incidence of healing at 12 weeks was 71% in the Oasis group versus 46% in the SOC group. Fisher’s Exact Test yields a significant p-value of 0.018. These results clearly demonstrate that the healing of these hard-to-heal wounds is markedly improved when Oasis wound matrix is used as part of the wound treatment program as compared to standard of care alone.

Notes: Background: Over the last two decades, there have been a number of studies in Europe on contact sensitivity in patients with chronic leg ulcerations with a frequency of positive patch test results ranging from 40 to 82.5%. The prevalence of sensitization has not been studied in North America. Furthermore, many of the newer dressings and wound care products in the market have not been studied for contact sensitivity in patients with chronic wounds. Objectives: 1) To determine the prevalence of allergen sensitivity in patients with history of leg ulcers in two North American study centers, 2) to compare our results to the European studies and to the North American Contact Dermatitis Group (NACDG) database and 3) to help delineate a standard battery of allergens for patch testing in North American leg ulcer patients Methods: 54 patients with an active or past leg ulcer were prospectively entered in the study. The patients were patch tested to both the NACDG Standard series, as well as, a comprehensive supplemental series of 48 allergens including wound care medicaments and dressings. Results: 63% of patients were sensitized to at last one allergen. The most common allergens were Balsam of Peru (29.6%), bacitracin (24.1%), fragrance mix (20.4%), wood tar mix (20.4%), propylene glycol (13.5%), neomycin sulfate (13%), benzalkonium chloride (13%), carba mix (11.1%), nickel sulfate (11.1%) and Duoderm CGF (11.1%). Duoderm CGF was the most allergenic dressing in our study group. Conclusion: There is a high incidence of positive patch tests in patients with past or current leg ulcerations. Using a modified leg ulcer series along with the standard NACDG series is very important in evaluating patients with leg ulcers.

Notes: Although electrical stimulation (ES) has been successful in the treatment of various types of wounds, the popularity of the treatment has waned due to a lack of optimal methods of administration. The goal of this study has been to develop and evaluate a method of delivering ES that is easy to use and suitable for delivery in a home-health environment.[0] Specifically, a novel bandage system developed to provide an electric field in a manner resembling the natural wound current was tested in vivo. In this study, a full-thickness wound model in New Zealand white rabbits was used to measure the effects of such a bandage system on healing of [0]skin defects. Different levels of current were evaluated initially, with 50 and 20 μA selected for the focus of this study, along with a non-stimulated control. Using histomorphometry healing rates, cellularity, and blood vessels were quantified at one and two week time points.The study showed the ability of this ES bandage to speed the healing process. An increase in overall healing rate over non-stimulated wounds of 45%(p = 0.04) was observed in wounds stimulated with 50 μA of current for 1 week. In wounds treated for 2 weeks, contraction rate decreased as well as the ratio of contraction rate to epithelialization rate. The stimulated wounds also showed an increase in the amount of macrophages, with a 79% increase in number of macrophages in wounds stimulated with 20 μA for 2 weeks and a 198% increase in macrophages in those stimulated with 50 μA compared to non-stimulated wounds. The performance of the bandage in the animal model has led to a limited clinical study on pressure ulcer patients using the 50 μA system. Acknowledgements: This project was funded through Biofisica and the CDC through NCIPC.

Notes: There have been many advances in the treatment of wounds made in the last decade. Innovative techniques of wound closure, topical agents, aggressive vascular repair, focused wound care management, and adjunctive hyperbaric oxygen therapy are but a few of these improvements. The vital role of oxygen in wound healing is becoming better understood, in no small part, due to Dr. T. K. Hunt and his colleagues at the Wound Healing Laboratory at the University of California, San Francisco. Elements of that contribution will be examined in this article. How these elements may be applied to improve wound healing will be explained and the possible role of adjunctive hyperbaric oxygen therapy based on sound science in the management of the difficult diabetic foot wound, will be highlighted.

Notes: Lens regeneration in adult newts is always initiated from the dorsal iris by transdifferentiation of the pigment epithelial cells. One of the most important early events should be the ability of pigment epithelial cells to dedifferentiate and re-enter the cell cycle. As a first step in an attempt to study this event, we have decided to examine the effects of a cyclin-dependent kinase-2 inhibitor on lens regeneration. At the appropriate concentration, this inhibitor completely abolished the ability of pigment epithelial cells to form a new lens, but it did not stop them from dedifferentiating and forming a small lens vesicle. The effects of this inhibitor seem to be mediated by its opposite effects on cell proliferation and apoptosis. The inhibitor significantly reduced cell proliferation and enhanced apoptosis of pigment epithelial cells both in vitro and in vivo and of the regenerating lens in vivo.

Notes: Cytokine growth factor treatment of chronic wounds has met with mixed results. The chronic wound presents a hostile environment to peptides such as growth factors. Cytokine growth factors have not been studied extensively in acute wounds. However, incisional hernias are a major example of acute wound failure that has not been solved by various mechanical approaches. A biological approach to acute wound failure by use of cytokine growth factors may offer a new strategy. A rodent incisional hernia model was used. Seventy-six rats underwent 3-cm midline celiotomies and were closed with fine, fast-absorbing sutures to induce intentional acute wound failure. Group 1 received no other treatment. The midline fascia in Groups 2–10 was infiltrated with 100 µl of vehicle alone or vehicle containing various test cytokine growth factors. Necropsy was performed on postoperative day 28 and the wounds were examined for herniation. Incisional hernias developed in 83 percent (13/16) of untreated incisional and 88 percent (7/8) and 83 percent (5/6) of the two vehicle-treated incisions (PBS and carboxymethylcellulose). Hernia incidences were decreased by priming of the fascial incision with transforming growth factor-β2 (12%, 1/8), basic fibroblast growth factor (25%, 2/8) and interleukin-1β (50%, 3/6) (p 〈 0.05). Aqueous platelet-derived growth factor, becaplermin, insulin-like growth factor, and granulocyte macrophage-colony stimulating factor did not significantly decrease the incidence of acute wound failure (p 〉 0.05). A biological approach to acute wound failure as measured by incisional hernia formation can be useful in reducing the incidence of this complication. Transforming growth factor-β2, basic fibroblast growth factor, and interleukin 1β all eliminated or significantly reduced the development of incisional hernias in the rat model.

Notes: Patients with diabetic neuropathy have reduced numbers of cutaneous nerves, which may contribute to an increased incidence of nonhealing wounds. Nerve growth factor (NGF) has been reported to augment wound closure. We hypothesized that topical 2.5S NGF, a biologically active subunit of the NGF polymer, would accelerate wound repair, augment nerve regeneration, and increase inflammation in excisional wounds in diabetic mice. A full-thickness 6-mm punch biopsy wound was created on the dorsum of C57BL/6 J-m + Leprdb mice (db/db) and heterozygous (db/–) littermates and treated daily with normal saline or 2.5S NGF (1 µg/day or 10 µg/day) on post-injury days 0–6. Time to closure, wound epithelialization, and degree of inflammation were compared using a Student's t-test. Color subtractive-computer-assisted image analysis was used to quantify immunolocalized nerves in wounds. Non-overlapping (20×) digital images of the wound were analyzed for nerve profile counts, area density (number of protein gene product 9.5 positive profiles per unit dermal area) and area fraction (protein gene product 9.5 positive area per unit dermal area). Healing times in db/db mice decreased from 30 days in normal saline-treated mice to 26 days in mice treated with 1 µg/day NGF (p 〈 0.05) and 24 days in mice treated with 10 µg/day NGF (p 〈 0.02). A similar trend in db/– mice was not significant. NGF treatment augmented epithelialization in the db/db mice (p 〈 0.05). Histological evaluation of inflammation in healed wounds showed no statistical difference between treatment groups. Total nerve number, area density, and area fraction were increased in NGF-treated wounds at 14, 21, and 35 days (p 〈 0.05). The 2.5 NGF subunit may improve wound closure kinetics by promoting epithelialization and nerve regeneration. Further studies to determine the role of nerves in wound repair are warranted.

Notes: Tissue engineering is an application for gene therapy that is in its infancy. We show that simple liposomal-mediated gene transfer could result in a potentially useful biological effect in the field of wound healing. cDNA encoding the 165 amino acid form of vascular endothelial growth factor complexed to commercially available liposomes was injected into rat skin 1 week before raising a random pattern 3 × 10 cm flap. The flap survival was enhanced by 14 percent, and was accomplished without accessing the arterial inflow of the territory. These results were statistically significant (p 〈 0.002) and reproducible. No adverse effects were seen. Histological analysis of the angiogenesis localized much of the new vessel formation to the area around the hair follicles. Polymerase chain reaction amplification of extracted flap tissue confirmed the presence of the transgene.

Notes: JCR:LA-cp/cp obese rats and their lean controls were evaluated as a type 2 diabetic wound healing model and the healing quality was characterized. This model of insulin resistance has been used extensively to study atherosclerosis but has not previously been used to study wound healing. Six circular excisional wounds were made on the dorsum of each rat and followed to day 21.Tracings of the wounds were made and used to assess the rate of wound closure. Planimetry showed a significantly diminished contraction of wounds in obese rats, but no significant difference in reepithelialization was observed. Collagen content was determined from the hydroxyproline content in wounded and unwounded skin. There were significantly lower levels of hydroxyproline in the wounds of obese compared to lean animals at day 21. Histology showed adipose tissue in place of dermal tissue in the JCR:LA-cp/cp rat in both unwounded tissue and in the wound at day 21. Active transforming growth factor-β1 (TGF-β1) was measured in the serum using the plasminogen activator inhibitor-1/luciferase assay and serum total TGF-β was measured using an enzyme-linked immunosorbent assay. Active TGF-β was significantly higher in the serum of obese animals compared with lean animals, while total TGF-β1 was not significantly different between the groups. Both active and total TGF-β was measured in tissue sections using the plasminogen activator inhibitor-1/luciferase assay. There was no significant difference in active TGF-β between genotypes, while obese rats had significantly higher levels of total TGF-β at day 21. These results indicate a deficiency in wound healing in obese animals characterized by decreased wound contraction, decreased collagen production, and changes in histology. The JCR:LA-cp rat develops insulin resistance, atherosclerosis and early type 2 diabetes and may be a good model for impairment of wound healing in humans with metabolic syndrome.

Notes: Cell proliferation and migration are important repair mechanisms in cell defect type mucosal injuries, such as peptic ulcers. To evaluate the level of cell restitution in vitro, we established a normalized assay system for analyzing the area of a tissue defect created in the center of a cultured cell layer. Although proton pump inhibitors are known to be potently effective in the treatment of peptic ulcers by inducing acid suppression, they are also effective in low-acid conditions, such as in gastric ulcers associated with severe atrophic gastritis of the corpus. The present study was designed to examine the pH-independent effect of lansoprazole (LPZ) on cell restitution in vitro. The mouse gastric mucosal cell line, GSM06, was cultured to confluence. A 4-fluoric ethylene-tipped aluminum stick was then used to produce a cell-free area in the center of the culture well. After measuring the area of the cell defect using a digital analyzer equipped with an inverted microscope, LPZ was added to each well; the area of the residual cell defect was then measured 6 and 24 hours after LPZ administration. To investigate the involvement of the p44/p42 mitogen-activated protein kinase (MAPK) and p38 MAPK in this process, PD98059 (a MEK inhibitor) or FR167653 (a p38 MAPK inhibitor) was added to the cell cultures. In a separate experiment, GSM06 cells were cultured to the subconfluent level, each test agent was added, and the cell number in each well was measured using an MTT assay 16 hours after the administration of the agents. Six hours after the addition of LPZ, a slight but significant increase in the cell restitution rate was observed in the LPZ-treated groups compared with that in the control group. After 24 hours, a further significant increase in the cell restitution rate was observed in the LPZ groups compared with that in the control group. While the addition of PD98059 significantly attenuated the cell restitution rate in the LPZ groups, the addition of FR167653 had no such effect. The total cell number in the subconfluent cell cultures was significantly increased in the LPZ-treated groups compared with that in the control group. In conclusion, LPZ promotes the healing of injured gastric mucosal cells following injury by enhancing cell proliferation and migration. Furthermore, the mechanism by which cell proliferation and migration is promoted by LPZ may involve the activation of p44/p42 MAPK.

Notes: Aim: To evaluate the efficacy of wound dressing material, Calcium-Zinc Alginate: Curasorb Zn® as a carrier of the recombinant human bFGF, histological detection was performed. Methods: Full thickness skin defect on the back of Wistar rat was made for four groups; the control group, bFGF group; topical application of recombinant human bFGF for 5 μg per day, Ca-Zn Alginate group; topical use of the dressing and the group that both the bFGF and Ca-Zn Alginate were applied, nine rats for respective group. Results: Significant granulation was noted both bFGF group and Ca-Zn Alginate group compared to the control group. Fibrosis was noted in the control and Ca-Zn Alginate group whereas no fibrosis was noted in the FGF group. Numerous amounts of cells were noted in the FGF group which indicate strong inflammation. Also, marked abscess formation was noted in the bFGF group. The greatest thickness of granulation was obtained in the group that both the bFGF and Ca-Zn Alginate were applied. Conclusions: It was considered that the appropriate use of the carrier for the bFGF is important to control infection. The Ca-Zn Alginate dressing was effective to suppress abscess formation.

Notes: Aim: It is reported that Granulocyte colony-stimulating factor (G-CSF) increases the number and functions of the neutrophils of blood in animal models. In the present study, we observed the effects of G-CSF in rats with severe acute pancreatitis. Methods: Pancreatitis was induced by injection of 0.2 ml of 3% taurocholate acid into biliopancreatic duct of the Wistar rat. Thirty rats were randomized into three groups. 1: Control group (C group). 2: Acute pancreatitis (AP) group. 3: AP + G-CSF groups. G-CSF was administrated via jugular veins 1 hour before induction of pancreatitis. Blood and ascites at 1 and 3 hours after induction of pancreatitis were measured for the number of neutrophils, their phagocytosis and bactericidal activity and the concentrations of TNF-á, IL-6 and IL-1â. Results: The phagocytic neutrophils increased in AP + G-CSF group (22 ± 4.3 × 105) compared with AP group (10 ± 1.9 × 105)(p 〈 0.05); the bactericidal neutrophils increased in AP + G-CSF group (60 ± 14.5 × 105) compared with AP group (20 ± 6.1 × 105)(p 〈 0.05). G-CSF did not increase any the concentration of TNF-á, IL-6 and IL-1â in blood and ascites. Conclusions: G-CSF increases the number of phagocytic and bactericidal neutrophils of blood and ascites in SAP rat, without increasing the concentration of TNF-á, IL-6 and IL-1â, therefore improves host defense against infection.

Notes: Introduction: It is reported on 3 cases of pressure ulcer which were hard to cure surgically by flap plastic operation or else, were completely healed by concomitant use of the basic fibroblast growth factor (bFGF) and the dressing. Case report: CASE 1:A 55-year-old male with a pressure ulcer 3 cm in diameter formed on the buttock. After excising the margin and spraying bFGF, the wound was covered with a dressing made of hydrofiber. It was completely cured in 3 months. CASE 2:A 65-year-old male with a pressure ulcer on the sacral region 30 cm the major axis. The pocket was cut open, and bFGF was sprayed inside. Amounts of the dressing material depended on volumes of the exudate. It was cured completely in 6 months. CASE 3:A 85-year-old male with a pressure ulcer 10 cm in the major axis on the greater trochanteric region of the right side. The margin was excised to open the pocket, bFGF was sprayed, and hydrogel was used as the dressing. It was cured in 6 months completely. Discussion: Usefulness of the conservative treatment is reported based on the experience referring to these 3 cases with pressure ulcer, which were completely cured upon concomitant use of bFGF and the dressing material suitable to the conditions of the wound after thorough surgical debridement and washings.

Notes: A 72-year-old female, who had slight impaired glucose tolerance, was diagnosed the second stage of carcinoma of the vulva. On the second day after radical vulvectomy with inguinal lymphadenectomy, the center of the wound changed color into deep purple. On the 8th postoperative days, the swelling of the wound extended over the mons pubis and a thigh, so the infected wound, which reached the epimysium, was opened for the debridement. Pseudomonas aeruginosa and gram negative bacilli were detected from the wound. Firstly, the wound was cleaned by a physiological salt solution, streptokinase, and sulfadiazine silver. The gynecologist and nurses in charge discussed about the therapy under the consultation of a dermatologist and treated the wound by the wet treatment that a piece of gauze moisten by a physiological salt solution was exchanged 3 or 4 times a day after disinfecting by povidone iodine. On the13th postoperative days, the infection was controlled and on the19th days the skin was grafted from a thigh. After the successful skin grafting the patient was discharged. Carcinoma of the vulva tends to occur in elder women. Wounds are often dissociated because of an interruption in the circulation by the excessive extension of the skin. From the successful experience of wet treatment after a surgical wound infection, we recognized the necessity of close observation on surgical site and the importance of cooperation of the doctors and the nurses in charge.

Notes: Introduction: Mediastinitis, which is at times encountered after thoracic surgery with median sternotomy, is often resistant for treatments and in cases leads to death. We applied vacuum-assisted closure (V.A.C.) for 2 cases of post-sternotomy mediastinitis, in an original manner using readily available materials and made them healed. The details are reported. Methods: We applied polyurethane foam (HydroSite®, Smith & Nephew) with silicone tube (BLAKE®, ETHICON) on the debrided wound, covered it with surgical draping film (Ioban® 2, 3M Health Care), and applied continuous negative pressure with a wall suction. The dressing was changed at intervals of 2 to 3 days. Results:(Case 1) A 53-years old male with complications of diabetes mellitus and hypertension underwent coronal artery bypass graft surgery for angina pectoris. Sternal infection occurred in postoperative period and the wound was opened. (Case 2) A 78-years old male underwent total aortic arch replacement surgery for aortic dissection. The wound was opened for a postoperative infection. We applied vacuum-assisted closure therapy for these 2 cases in the method presented above and observed promoted wound granulation and rapid contraction of wounds, with no manifestations of increasing infection or changes of circular conditions. Each of the cases was healed with vacuum-assisted closure in 7 Weeks without any surgical procedures. Conclusions: We viewed vacuum-assisted closure therapy as another choice of the treatment for mediastinitis.

Notes: Aim: In tissue engineering of the skin, the selection of a scaffold or a matrix in which a cultured cells grow is quite important. We have developed a tissue engineering skin composed of human keratinocytes and fibroblasts on an acellular allogenic dermal matrix (ADM), derived from cryopreserved human skin. Methods: ADM was prepared from cryopreserved split-thickness human skin by treating with Dispase and Triton X-100. The tissue engineering skin were produced by seeding human keratinocytes and fibroblasts on ADM. Several days after seeding, the tissue engineering skin was exposed to an air-liquid interface for another 7 days. Then, the histological structure of the skin and the production of growth factors by the skin were investigated. Results: The produced ADM was found to be completely acellular with remaining structure of the basement membrane components, such as type IV collagen and laminin. The developed tissue engineering skin had stratified keratinocytes on the surface of the ADM migrating fibroblasts in the dermal collagen structure, resembling to the normal skin appearance. It was found that several important growth factors in wound healing process, such as TGF-α, TGF-β and VEGF, were produced by the tissue engineering skin. Conclusions: It was suggested that ADM is suitable for a scaffold in tissue engineering of the skin.

Notes: This study was designed to try the application of autologous cultured dermal substitute (CDS) in conjunction with patient's own epidermis. The autologous CDS was prepared by seeding cultured autolougus fibroblasts on the spongy matrix of hyaluronic acid and atelo-collagen. A 9-year-old man with a giant congenital pigmented nevus (intradermal type) was included in this clinical study. A part of nevus (30 cm × 10 cm) was excised superficially at a thickness of 20/1000 inches, and followed by second excision to remove nevus at a level of full-thickness skin. The autologous CDS was applied to the debrided wound surface. The split-thickenss skin obtained by first excision was preserved at 4 °C. After 1 week, the epidermis was obtained from the preserved split-thickness skin using dispase, and followed by grafting on the wound bed, which was prepared by applying autologous CDS. This patient's own epidermis was found to take permanently, achieving an excellent clinical results.

Notes: Aim: A treatment of extensive burn contracture in children needs to repeat the autologous skin graft. This study was designed to evaluate the application of autologous CDS to prepare the proper wound bed acceptable for the split-thickness autologous skin graft. Methods: Prior to the clinical study, the master cell banking system was established using a small piece of skin derived from each patient. The autologous CDS was prepared by plating the patient's own fibroblasts, cultured from the master cells, on a spongy matrix of hyaluronic acid and atelo-collagen. The CDS was applied on the skin defects left behind surgical excision to release scar contracture. The split-thickness autologous skin graft (6∼8/1000 inch) was applied on the wound bed, prepared by using the CDS. Results: The clinical trials were conducted in 5 cases. When the autologous CDS was applied on the skin defect, exposing subcutaneous fatty tissue, the highly vascularlized wound bed was prepared within about 1 week. Although a split-thickness skin graft was very thin, the severe contracture was not observed over a period of several months. Conclusion: The application of autologous CDS is promising for the treatment for extensive burn scar contracture in children.

Notes: Aim: Clinical researches using allogeneic CDS, developed in the R & D Center for Artificial Skin of Kitasato University, have been carried out in 30 medical centers across Japan. The clinical results in our hospital, especially focusing on the treatment of refractory ulcers and dermal burns, were reported in this study. Methods: The CDS was prepared by plating cultured fibroblasts on a spongy matrix of hyaluronic acid and atelo-collagen. The CDS was used in 13 clinical trials, including 6 refractory ulcers, 4 dermal burns, 1 skin defect left behind preparing skin flaps, and 2 skin defects left behind removal of scar. The use of CDS in conjunction with a conventional ointment-gauze dressing was repeated at an interval of 4 to 7 days over a period of 2 to 6 weeks. Results: The successful application with CDS was achieved in all cases. Especially, in case of refractory ulcers, failed to heal even by using trafermin (Fiblast spray®) or other ointments, a complete healing was achieved in one case and wound size reduction was observed in other 5 cases within 6 weeks. Conclusions: Our results suggest that the CDS is effective in the treatment of refractory ulcers and other skin defects. The excellent clinical results seem to be related to the cytokines released from the CDS.

Notes: We have been cultivating human epidermal cells for therapeutic purpose according to the original methods developed by Rheinwald and Green. Cultured epithelium (CE) was applied to patients with severe skin defects, burn wounds, chronic skin ulcers and cutaneous disorders like hypomelanosis. Autologous CE allows to restore massive skin surface in a short period compared with other conventional treatments. For grafts take, it is important to manage wound beds properly prior to CE grafting. The CDS was applied to prepare wound bed acceptable for CE grafting. The CDS was designed to secrete various types of cytokines, i.e., VEGF and KGF to stimulate wound healing. The successful management of deep wounds like chronic skin ulcer or burn ulcer requires granulation tissue formation and epithelialization to wound closure. This study aimed to evaluate the application of CDS in conjunction with CE for patients with chronic skin ulcer and burn ulcer. In some cases the wounds were cured by using CDS, and followed by CE grafting. All clinical trials achieved excellent or good results, showing no contracture and no hypertrophic scar after wound closure. The CDS was found to be useful to prepare wound beds and to facilitate wound management.

Notes: Aim: Keloid lesions develope as a result of abnormal growth of dermal fibroblasts after the injury. On the other hand, cell division cycle (Cdc) 25 is a family of phosphatases that activate the cell cycle regulating cyclin-dependent kinases. The three members of this family, Cdc25A, Cdc25B, and Cdc25C act in different phases of the cell cycle. In this study, we examined the dgree of expression of these phosphatases in keloid fibroblasts. Methods: Primary cultures of keloid and adjacent normal dermal fibroblasts (n = 4) as well as frozen and paraffin-embedded keloid and normal dermal tissues (n = 12 and n = 17 respectively) were examined by Western blot and immunohistochemical analyses for the expressions of Cdc25A, Cdc25B, Cdc25C and phosphorylated Cdk2. Results: Cdc25A protein levels were frequently increased in keloid fibroblasts as compared to the adjacent normal-appearing fibroblasts or different normal dermal fibroblasts. In contrast, Cdc25B and Cdc25C were none or rarely expressed. The incrased levels of Cdc25A were associated with lower levels of its substrate, Cdk2, in a phosphorylated or inactive form. Conclusions: Taken together, our data show that Cdc25A protein levels increase in keloid fibroblasts and this increase may be sufficient to activate its substrate, Cdk2, and accelerate the cell cycling. These results may have implication for the development of strategies to silence Cdc25A activity after the wound healing as a therapeutic modality for the keloid lesions.

Notes: The present immunohistochemical study was designed to investigate the manner of the myenteric nerve regeneration and expression of glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor (Ret) in nerves after transection of the muscle coat in the rat small intestine. The enteric neurons and enteroglial cells were immunohistochemically determined using antibodies against protein gene product 9.5 and S-100 protein, respectively. The neuronal sprouts issued from the severed nerve stumps 12 h after the operation, and thereafter extended into the lesion. The proximal portions of outgrowing neuronal fibers were gradually enveloped by regenerating enteroglial cells to become thick nerve bundles. The regrowing neurons and their associated enteroglial cells developed into an irregular network in the myectomized area on postoperative day 5. The elongation of the regrowing nerves was conspicuously accelerated from postoperative day 3 in accordance with the regrowing neurons started to associate with the enteroglial cells. Under normal conditions, faint immunoreactivity for GDNF and Ret was selectively localized in the enteroglial cells and neurons within the myenteric ganglia, respectively. Following myectomy, the both immunoreactivities were significantly intensified in the nerve stumps and ganglia proximal to the lesion. The regenerating enteroglial cells closely associated with the regrowing neurons in the lesion exhibiting a dense immunoreaction to GDNF, whereas the neuronal fibers were richly supplied with reaction products of Ret in their entire course. The present findings suggest that the enteroglial cells, contacting with regrowing neurons, may promote the myenteric nerve regeneration in the rat small intestine, via the GDNF-Ret signaling system.

Notes: Aim: Quality of Life (QOL) of patients after cancer surgery has become more important as success rate of surgical treatment has been improved. Anal sphincter preservation has been concerned in treatment for colon cancer, in particular rectum cancer. However, in order for patients to become cancer free, colostomy is the essential treatment. In usual stoma, skin contacts directly with colon membrane, and this sometimes induces (i) skin troubles around the stoma (e.g., ulcer, abscess, fistula), and (ii) constriction, collapse, perforation, hernia or obstruction of the colon. The skin troubles are the complications that are unavoidable only through the improvement and control of the equipment because the skin directly touches to the stool. Therefore, preventive measures should be employed at colostomy. For this purpose, a new colostomy technique was applied from the point of plastic and reconstruction surgery. Methods: In order to reduce the duration and area of direct contact with stool, local flap and split-thickness skin grafting are applied. Colon is pull out to the abdominal wall by using the usual surgical procedure. The stoma is kept at a certain height from the abdominal wall by using the flap and the graft. Results: To date, the longest follow-up period is 8 years and 7 months. Frequency of skin problems due to direct contact with stool was reduced, infection and constriction on and around the stoma have been prevented, and defecation became able to be better managed.

Notes: Aim: The movements of the cell population are different between 2-D culture and 3-D culture. From the observation of morphology of skeletal muscle cell 3-D culture, We expect that skeletal muscle cells differentiation is accelerate in the collagen gel 3-D culture, and the proliferation is suppressed. The purpose of this study is to investigate the difference between 2-D culture and 3-D culture of C2C12 cells. Methods: C2C12 skeletal muscle cells are incubated following three difference conditions for 48 hours, plastic dish 2-D culture, collagen coated dish 2-D culture and collagen gel 3-D culture. The culture medium is Dulbecco's modified Eagle medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Collagens are removed by collagenase treatment and cells are homogenized. After centrifugation the top clear layer is used for CPK assay and protein development analysis by Western blotting Results: After 48 hour incubation, we observed cell morphology by a phase contract microscope. Cell fusion was observed in collagen gel 3-D culture. The fusion cells have many nucleus in the cytoplasm called synthetium. But in plastic dish 2-D culture and in collagen coated dish 2-D culture synthetiums were not observed and cells were mononuclear and monolayer. Cell prolieration was suppressed in collagen gel 3-D culture. CPK activity was five times activated in collagen gel 3-D culture than in plastic dish 2-D culture. Conclusions: We suggest skeletal muscle cells C2C12 are activate differentiation by collagen gel 3-D culture.

Notes: Abstract: On 11 patients with skin defect and refractory ulcer, treatment was conducted using bFGF preparations. Good granulation, epithelialization and reduction of ulcer were observed in all patients by once-daily spraying of this product after washing the topical region. This preparation is a drug developed aiming at the basic fibroblast-proliferating factor playing an important role in the wound healing process in the body and expecting the therapeutic effects on refractory skin ulcer. Although there are some restrictions including the tropical conditions such as infection and adherence of necrosed tissues and careful administration to the patients with a past history of malignant tumor, its good therapeutic effects were expected in the patients ineffective with other drugs, and this product was evaluated to be useful from the viewpoint of QOL, such as shortening of treatment period, reduction of cost and low invation.

Notes: Aim: A method for preparing acellular trachea scaffold and its effectiveness were investigated by using rabbits and dogs Methods: The sacrificed dog or rabbit trachea was collected. The spiral stent of stainless steel was inserted in the obtained trachea. To remove all of cellular components in tissues, the trachea was rinsed with sterilized 0.5% Triton X-100 for 24 to 48 hours at ambient temperature and then for removing the detergents completely using fresh water. The acellular trachea obtained was lyophilized and sterilized by ethylene oxide gas. Before implantation, the lyophilized acellular trachea was soaked in phosphate buffered saline containing 0.5% gelatin and other adhesive molecules for 2 to 18 hours at 37 degree. After 15 mm of rabbit neck trachea was removed surgically under anesthesia, the same length of reengorged acellular rabbit trachea was implanted the removed region. In the case of dog, 50 mm of thoracic trachea was removed under the mechanical ventilation and then the same length of reengorged acellular trachea was implanted by technique of end to end anastomosis. The implanted trachea was rapped by omentum. The effectiveness of acellualr scaffold on implanted-animals was evaluated by endoscope finding. Results: 1) The rabbits implanted reengorged-acellular trachea survived for minimum 10 days and maximum 60 days. It was suggested that the cause of death was the infection of implantation region. 2) The dog implanted reengorged-acellular trachea survived for over 60 days at least. The cause of death was strangulated hernia. 3) The acellular trachea containing various growth factors or cultured with fibroblasts was not always effective. Discussion: It is not necessary for the animal implanted acellular trachea to be administered the immunodepressants such as the animal implanted cryopreserved trachea. From these results, the tissue engineered acellular trachea may be more effective than the cryopreserved trachea.

Notes: Incurable ulcer in the lower legs means avascular skin defects exposing bone and osteomyelitis which cannot be repaired even with skin grafting. A total of 151 patients with incurable ulcers were operated with flaps; 60 cases of traumatized avascular defects, 20 diabetic ulcers, 17 osteomyelitis, 14 malignant tumors, 5 arterial obstructions, and 5 arteriovenous malformations, and others. A total of 61 island flaps were used; 20 posterior tibial perforator flaps, 7 saphenous flaps, 7 peroneal flaps, 4 anterior tibial flaps, 4 malleolar perforator flaps, 4 medialis pedis flaps, and others. In addition, a total of 82 free flaps using microvascualr anastomosis were sued; 24 flow-through anterior thigh flaps, 13 flow-throuh thoracodorsal artery perforator flaps (or latissimus dorsi MC flap), 8 paraumbilical (or deep inferior epigastric artery ) perforator flap, 6 saphenous venous flaps, 7 combined flaps, and 24 others. In conclusions, small ulcers could be repaired with minimal invasive methods including local perforator flaps and small muscle flaps under local anesthesia. Free flow-through flaps and free bypass flaps (for diabetic gangrene with ASO), and combined osteocutaneous flaps (for massive segmental defects after resecting advanced carcinoma) are indicated for large ischemic defects.

Notes: The combined application of cytokines on embryonic fibroblasts and dermal substitute were extensively studied for optimal skin defect coverage. Signalling of combined treatment of leukemia inhibitory factor (LIF) and vascular endothelial factor (VEGF) were elucidated and subsequently the in vivo applications of both were tested in an artificial dermal substitute. Mouse embryonic fibroblast cells, BALB-3T3, were stably transfected with mouse full length LIF cDNA and added to various doses of VEGF for detection of signalling interaction. LIF-transfected cells and VEGF treatment were tested with pig-tendon derived collagen dermal substitute in the backs of BALB/c male mice for 14 days. LIF-transfected cells as well as vector-transfected fibroblasts significantly proliferated by 1, 10, or 100 nM VEGF on days 3 and 5. LIF-transfected cells showed rapid phosphorylation of STAT 3 from 1 minute to 60 minutes after VEGF treatment, while vector-transfected cells failed to induce such phosphorylation after VEGF treatment. Erk MAP kinase phosphorylation was observed from 1 to 15 minutes in LIF-transfected and 10 nM of VEFG and 1 to 30 minutes in LIF-transfected and 100 nM VEFG treatments. In in vivo analyses, LIF-transfected embryonic fibroblasts with 50 μg of VEGF markedly enhanced collagen I expression and CD 34 angiogenic marker on days 7 and 14. LIF transfection induced constitutive STAT signaling and enhanced phosphorylated-Erk MAP kinase with exogenous VEGF. In vivo study revealed that the combined application of LIF-transfection of embryonic fibroblasts with an angiogenic factor such as VEGF in the template of an artificial dermis.

Notes: Aim: Various treatments for hypertrophic and keloid scars have been attempted including electron beam irradiation, local triamcinolone injection, oral tranilast administration, use of silicone sheets, and compression therapy using splints. Among them, we have been attempting silicone cushion patching on hypertrophic scar. Methods: Twenty cases (10 males, 10 females) with hypertrophic scar were treated with silicone cushion. Scar surfaces were kept in contact with silicone cushion for as long periods of time as possible every day. Results were assessed in scores using objective findings (redness, bulging, induration) and subjective symptoms (itching, spontaneous pain, tenderness). Results: As for objective findings, redness remained unchanged in one case, but bulging and induration were found improved in all cases. As for subjective symptoms, itching and tenderness were found improved in all cases and clinical course considered proving usefulness of the treatment was obtained. Conclusions: Silicone cushions have highly viscous silicone oil enclosed in the silicone pack designed to generate negative charge electrostatic fields. Generation of adequate and sustaining electrostatic fields is important for useful clinical effect to exhibit on hypertrophic scar recession. Generation of negative charge electrostatic fields was confirmed on potential determination data with an electrostatic field meter as well.

Notes: Aim: When surgery with postoperative superficial electron irradiation is applied, the recurrence rate of keloid lesion has been found to be 〈30%. In this study, we assessed the molecular changes underlying the effect of electron irradiation by differential global gene expression analyses of both cultured keloid and normal skin fibroblasts. Materials and Methods: Primary cultured fibroblasts from 4 active keloids and their adjacent normal dermal tissues were irradiated at a calibrated dosage of 15 Gy with 6 MeV electron beam generated by a linear accelerator. Corresponding paired non-irradiated cells were used as control. RNA isolated from the collected cells was labeled with 33P, hybridized to the cDNA microarray gene filters and analyzed. Results: After irradiation, the gene expression profiles of keloid and normal skin fibroblasts were closely similar. Electron irradiated keloid fibroblasts showed suppressed levels of collagen typeI (alpha2), collagen typeVI (alpha1), matrix metalloproteinase 2, fibronectin 1, insulin-like growth factor binding protein 3, alpha-1-antichymotorypsin and heparan glucosaminyl 1 as compared with their non–irradiated counterparts. Conclusions: Downregulation of matrix synthesis and upregulation of protease inhibitors and apoptosis promoting genes by electron irradiation may inhibit keloid development. This mode of therapy appears to exert a positive effect toward lowering the recurrence rate of keloid formation.

Notes: Introduction: Oral mucosa heals faster with less scar than skin, but its mechanism has not been elucidated enough. We have studied whether there are any differences in the dynamic study of the bFGF expression in vivo during wound healing between oral mucosa and skin. Methods: Male Wistar rats were anesthetized, and a 3 mm circular exisional wound was made on the dorsum skin and intrabuccal mucosa of each rat. At days 0,1,3,5,7 and 10 post-wounding,wounds were harvested, and embedded in OCT. From photographs of the wounds, each wound area was measured by means of NIH images. Also, for histological study, sections were stained haematoxylin and eosin, and for immunohistochemical study, sections were stained with anti-bFGF antibody. Results: Oral wounds contracted significantly faster than skin. Regenerated skin appeared at day 1 post-wounding in oral wounds, but at day 3 in skin. Re-epithelialization was completed on day 5 post-wounding in oral, but on day 7 in skin. The number of bFGF-positive cells gradually increased in the granulation tissue, and the peak of positive cells in oral was observed on day 5 post-wounding, while that in skin on day 7. Conclusion: Oral mucosa healed faster than skin. The peak of bFGF expression was faster in oral mucosa than skin. It was thougt that bFGF effects the differences during wound healing between oral mucosa and skin.

Notes: Enamel matrix derivative, obtained from developing porcine teeth, is composed mainly of amelogenin proteins and used topically in periodontal surgery for advanced periodontitis to regenerate lost connective tissues. The primary objective of this study was to investigate the effects of enamel matrix derivative on skin wound healing. Secondly, in vitro effects of enamel matrix derivative on dermal fibroblasts and microvascular endothelial cells were examined. Full-thickness, circular 2-cm skin wounds in white 16-week-old rabbits were treated thrice weekly with enamel matrix derivative (30 mg/ml) in the vehicle propylene glycol alginate or with vehicle alone. Enamel matrix derivative treatment increased the amount of granulation tissue and accelerated time to complete epithelialization by 3 days (ρ 〈 0.001) compared to vehicle treatment. In cultured fibroblasts, vascular endothelial growth factor levels in conditioned media were increased more than fivefold (ρ 〈 0.001) with enamel matrix derivative treatment (0.1 mg/ml) over control, measured by specific enzyme-linked immunosorbent assay. Enamel matrix derivative also increased release of matrix metalloproteinase-2 more than threefold from fibroblasts (ρ 〈 0.001) and from endothelial cells (ρ 〈 0.001). Thus, enamel matrix derivative significantly accelerated wound closure in rabbits, possibly by increasing levels of growth factors and proteinases important for granulation tissue formation and remodeling.

Notes: Andalusite porphyroblasts are totally pseudomorphosed by margarite–paragonite aggregates in aluminous pelites containing the peak mineral assemblage andalusite, chlorite, chloritoid, margarite, paragonite, quartz ± garnet, in a NW Iberia contact area. Equilibria at low P–T are investigated using new KFMASH and (mainly) MnCNKFMASH grids constructed with Thermocalc 3.21. P–T and T–X pseudosections with phase modal volume isopleths are constructed for compositions relatively richer and poorer in andalusite to model the assemblages in an andalusite-bearing rock that contains a thin andalusite-rich band (ARB) during retrogression. Their compositions, prior to retrogression, are used in the modelling, and have been retrieved by restoring the pseudomorph-forming elements into the current-depleted matrix, except for Al2O3 which is assumed to be immobile. Compositional differences between the thin band and the rest of the rock have not resulted in differences in andalusite porphyroblast retrogression. The absence of chloritoid resorbtion implies either a pressure increase at constant reacting-system composition, or that its composition changed during retrogression at constant pressure, by becoming enriched in the progressively replaced andalusite porphyroblasts. T–X pseudosections at 1 kbar model this latter process using as end-members in X, first, the restored original rock and ARB compositions, and, then the same process, taking into account the change in composition of both as retrogression proceeded. The MnNCKFMASH pseudosections of rocks with different Al contents facilitate making further deductions on the rock-composition control of the resulting assemblages upon retrogression. Andalusite eventually disappears in relatively Al-poor rocks, resulting, as in this study, in a rock formed by chloritoid–chlorite as the only FM minerals, plus margarite–paragonite pseudomorphs of andalusite. In rocks richer in Al, chlorite would progressively disappear and a kyanite/andalusite–chloritoid assemblage would eventually be stable at retrograde conditions. The Al-silicate, stable during retrogression in Al-rich rocks, indicates pressure conditions and hence the tectonic context under which retrogression took place.

Notes: In statistically optimised P–T estimation, the contributions to overall uncertainty from different sources are represented by ellipses. One source, for a diffusion-controlled reaction at non-equilibrium, is diffusion modelling of the reaction texture. This modelling is used to estimate ratios, Q, between free-energy differences, ΔG, of reactions among mineral end-members, to replace the equilibrium condition ΔG = 0. The associated uncertainty is compared with those already inherent in the equilibrium case (from end-member data, activity models and mineral compositions). A compact matrix formulation is introduced for activity coefficients, and their partial derivatives governing error propagation. The non-equilibrium example studied is a corona reaction with the assemblage Grt–Opx–Cpx–Pl–Qtz. Two garnet compositions are used, from opposite sides of the corona. In one of them, affected by post-reaction Fe, Mg exchange with pyroxene, the problem of reconstructing the original composition is overcome by direct use of ratios between chemical-potential differences, given by the diffusion modelling. The number of geothermobarometers in the optimisation is limited by near-degeneracies. Their weightings are affected by strong correlations among Q ratios. Uncertainty from diffusion modelling is not large in comparison with other sources. Overall precision is limited mainly by uncertainties in activity models. Hypothetical equilibrium P–T are also estimated for both garnet compositions. By this approach, departure from equilibrium can be measured, with statistical uncertainties. For the example, the result for difference from equilibrium pressure is 1.2 ± 0.7 kbar.

Notes: This paper characterizes the metamorphic thermal structure of the Higo Metamorphic Complex (HMC) and presents the results of a numerical simulation of a geotherm with melt migration and solidification. Reconstruction of the geological and metamorphic structure shows that the HMC initially had a simple thermal structure where metamorphic temperatures and pressures increased towards apparent lower structural levels. Subsequently, this initial thermal structure has been collapsed by E–W and NNE–SSW trending high-angle faults. Pressure and temperature conditions using the analysis of mineral assemblages and thermobarometry define a metamorphic field P–T array that may be divided into two segments: the array at apparent higher structural levels has a low-dP/dT slope, whereas that at apparent lower structural levels has a high-dP/dT slope. This composite array cannot be explained by heat conduction in subsolidus rocks alone. Migmatite is exposed pervasively at apparent lower structural levels, but large syn-metamorphic plutons are absent at the levels exposed in the HMC. Transport and solidification of melt within migmatite is a potential mechanism to generate the composite array. Thermal modelling of a geotherm with melt migration and solidification shows that the composite thermal structure may be formed by a change of the dominant heat transfer from an advective regime to a conduction regime with decreasing depth. The model also predicts that strata beneath the crossing point will consist of high-grade solid metamorphic rocks and solidified melt products, such as migmatite. This prediction is consistent with the observation that migmatite was associated with the very high-dP/dT slope. The melt migration model is able to generate the very high-dP/dT segment due to the high rate of heat transfer by advection.

Notes: This study analyses the mineralogical and chemical transformations associated with an Alpine shear zone in polymetamorphic metapelites from the Monte Rosa nappe in the upper Val Loranco (N-Italy). In the shear zone, the pre-Alpine assemblage plagioclase + biotite + kyanite is replaced by the assemblage garnet + phengite + paragonite at eclogite facies conditions of about 650 °C at 12.5 kbar. Outside the shear zone, only minute progress of the same metamorphic reaction was attained during the Alpine metamorphic overprint and the pre-Alpine mineral assemblage is largely preserved. Textures of incomplete reaction, such as garnet rims at former grain contacts between pre-existing plagioclase and biotite, are preserved in the country rocks of the shear zone. Reaction textures and phase relations indicate that the Alpine metamorphic overprint occurred under largely anhydrous conditions in low strain domains. In contrast, the mineralogical changes and phase equilibrium diagrams indicate water saturation within the Alpine shear zones. Shear zone formation occurred at approximately constant volume but was associated with substantial gains in silica and losses in aluminium and potassium. Changes in mineral modes associated with chemical alteration and progressive deformation indicate that plagioclase, biotite and kyanite were not only consumed in the course of the garnet-and phengite-producing reactions, but were also dissolved ‘congruently’ during shear zone formation. A large fraction of the silica liberated by plagioclase, biotite and kyanite dissolution was immediately re-precipitated to form quartz, but the dissolved aluminium- and potassium-bearing species appear to have been stable in solution and were removed via the pore fluid. The reaction causes the localization of deformation by producing fine-grained white mica, which forms a mechanically weak aggregate.

Notes: Low-pressure crystal-liquid equilibria in pelitic compositions are important in the formation of low-pressure, high-temperature migmatites and in the crystallization of peraluminous leucogranites and S-type granites and their volcanic equivalents. This paper provides data from vapour-present melting of cordierite-bearing pelitic assemblages and augments published data from vapour-present and vapour-absent melting of peraluminous compositions, much of which is at higher pressures. Starting material for the experiments was a pelitic rock from Morton Pass, Wyoming, with the major assemblage quartz-K feldspar-biotite-cordierite, approximately in the system KFMASH. A greater range in starting materials was obtained by addition of quartz and sillimanite to aliquots of this rock. Sixty-one experiments were carried out in cold-seal apparatus at pressures of 1–3.5 kbar (particularly 2 kbar) and temperatures from 700 to 840 °C, with and without the addition of water. In the vapour-present liquidus relations at 2 kbar near the beginning of melting, the sequence of reactions with increasing temperature is: Qtz + Kfs + Crd + Sil + Spl + V = L; Qtz + Kfs + Crd + Spl + Ilm + V = Bt + L; and Qtz + Bt + V = Crd + Opx + Ilm + L. Vapour-absent melting starts at about 800 °C with a reaction of the form Qtz + Bt = Kfs + Crd + Opx + Ilm + L. Between approximately 1–3 kbar the congruent melting reaction is biotite-absent, and biotite is produced by incongruent melting, in contrast to higher-pressure equilibria. Low pressure melts from pelitic compositions are dominated by Qtz-Kfs-Crd. Glasses at 820–840 °C have calculated modes of approximately Qtz42Kfs46Crd12. Granites or granitic leucosomes with more than 10–15% cordierite should be suspected of containing residual cordierite. The low-pressure glasses are quite similar to the higher-pressure glasses from the literature. However, XMg increases from about 0.1–0.3 with increasing pressure from 1 to 10 kbar, and the low-temperature low-pressure glasses are the most Fe-rich of all the experimental glasses from pelitic compositions.

Notes: Quartz-rich veins in metapelitic schists of the Sanandaj-Sirjan belt, Hamadan region, Iran, commonly contain two Al2SiO5 polymorphs, and, more rarely, three coexisting Al2SiO5 polymorphs. In most andalusite and sillimanite schists, the types of polymorphs in veins correlate with Al2SiO5 polymorph(s) in the host rocks, although vein polymorphs are texturally and compositionally distinct from those in adjacent host rocks; e.g. vein andalusite is enriched in Fe2O3 relative to host rock andalusite. Low-grade rocks contain andalusite + quartz veins, medium-grade rocks contain andalusite + sillimanite + quartz ± plagioclase veins, and high-grade rocks contain sillimanite + quartz + plagioclase veins/leucosomes. Although most andalusite and sillimanite-bearing veins occur in host rocks that also contain Al2SiO5, kyanite-quartz veins crosscut rocks that lack Al2SiO5 (e.g. staurolite schist, granite). A quartz vein containing andalusite + kyanite + sillimanite + staurolite + muscovite occurs in andalusite–sillimanite host rocks. Textural relationships in this vein indicate the crystallization sequence andalusite to kyanite to sillimanite. This crystallization sequence conflicts with the observation that kyanite-quartz veins post-date andalusite–sillimanite veins and at least one intrusive phase of a granite that produced a low-pressure–high-temperature contact aureole; these relationships imply a sequence of andalusite to sillimanite to kyanite. Varying crystallization sequences for rocks in a largely coherent metamorphic belt can be explained by P–T paths of different rocks passing near (slightly above, slightly below) the Al2SiO5 triple point, and by overprinting of multiple metamorphic events in a terrane that evolved from a continental arc to a collisional orogen.

Notes: Zircon fission track dating and track length analysis in the high-grade part of the Asemigawa region of the Sanbagawa belt demonstrates a simple cooling history passing through the partial annealing zone at 63.2 ± 5.8 (2 σ) Ma. Combining this age with previous results of phengite and amphibole K–Ar and 40Ar/39Ar dating gives a cooling rate of between 6 and 13 °C Myr−1, which can be converted to a maximum exhumation rate of 0.7 mm year−1 using the known shape of the P–T path. This is an order of magnitude lower than the early part of the exhumation history. In contrast, zircon fission track analyses in the low-grade Oboke region show that this area has undergone a complex thermal history probably related to post-orogenic secondary reheating younger than c. 30 Ma. This event may correlate with the widespread igneous activity in south-west Japan around 15 Ma. The age of subduction-related metamorphism in the Oboke area is probably considerably older than the generally accepted range of 77–70 Ma.

Notes: Garnet-bearing ultramafic rocks including clinopyroxenite, wehrlite and websterite locally crop out in the Higashi-akaishi peridotite of the Besshi region in the Cretaceous Sanbagawa metamorphic belt. These rock types occur within dunite as lenses, boudins or layers with a thickness ranging from a few centimetres to 1 metre. The wide and systematic variation of bulk-rock composition and the overall layered structure imply that the ultramafic complex originated as a cumulate sequence. Garnet and other major silicates contain rare inclusions of edenitic amphibole, chlorite and magnetite, implying equilibrium at relatively low P–T conditions during prograde metamorphism. Orthopyroxene coexisting with garnet shows bell-shaped Al zoning with a continuous decrease of Al from the core towards the rim, consistent with rims recording peak metamorphic conditions. Estimated P–T conditions using core and rim compositions of orthopyroxene are 1.5–2.4 GPa/700–800 °C and 2.9–3.8 GPa/700–810 °C, respectively, implying a high P/T gradient (〉 3.1 GPa/100 °C) during prograde metamorphism. The presence of relatively low P–T conditions at an early stage of metamorphism and the steep P/T gradient together trace a concave upwards P–T path that shows increasing P/T with higher T, similar to P–T paths reported from other UHP metamorphic terranes. These results suggest either (1) down dragging of hydrated mantle cumulate parallel to the slab–wedge interface in the subduction zone by mechanical coupling with the subducting slab or (2) ocean floor metamorphism and/or serpentinization at early stage of subduction of oceanic lithosphere and ensuing HP–UHP prograde metamorphism.

Notes: During emplacement and cooling, the layered mafic–ultramafic Kettara intrusion (Jebilet, Morocco) underwent coeval effects of deformation and pervasive fluid infiltration at the scale of the intrusion. In the zones not affected by deformation, primary minerals (olivine, plagioclase, clinopyroxene) were partially or totally altered into Ca-amphibole, Mg-chlorite and CaAl-silicates. In the zones of active deformation (centimetre-scale shear zones), focused fluid flow transformed the metacumulates (peridotites and leucogabbros) into ultramylonites where insoluble primary minerals (ilmenite, spinel and apatite) persist in a Ca-amphibole-rich matrix. Mass-balance calculations indicate that shearing was accompanied by up to 200% volume gain; the ultramylonites being enriched in Si, Ca, Mg, and Fe, and depleted in Na and K. The gains in Ca and Mg and losses in Na and K are consistent with fluid flow in the direction of increasing temperature.When the intrusion had cooled to temperatures prevailing in the country rock (lower greenschist facies), deformation was still active along the shear zones. Intense intragranular fracturing in the shear zone walls and subsequent fluid infiltration allowed shear zones to thicken to metre-scale shear zones with time. The inner parts of the shear zones were transformed into chlorite-rich ultramylonites. In the shear zone walls, muscovite crystallized at the expense of Ca–Al silicates, while calcite and quartz were deposited in ‘en echelon’ veins. Mass-balance calculations indicate that formation of the chlorite-rich shear zones was accompanied by up to 60% volume loss near the centre of the shear zones; the ultramylonites being enriched in Fe and depleted in Si, Ca, Mg, Na and K while the shear zones walls are enriched in K and depleted in Ca and Si. The alteration observed in, and adjacent to the chlorite shear zones is consistent with an upward migrating regional fluid which flows laterally into the shear zone walls. Isotopic (Sr, O) signatures inferred for the fluid indicate it was deeply equilibrated with host lithologies.

Notes: As the best preserved high- and ultrahigh-pressure (HP and UHP) metamorphic terrane in the Qinling-Dabieshan-Sulu orogen, western Dabieshan is divided into six lithotectonic units along a traverse across the orogen, i.e. from north to south, the Nanwan, Balifan, Huwan, Xinxian, Hong'an and Mulanshan units. In this terrane five eclogite-bearing zones (I–V) are developed. The garnet and clinopyroxene in eclogites from these zones exhibit chemical zoning, suggesting that the rims record general peak temperature and pressure. Thermobarometric study indicates that the peak P–T conditions of eclogite are 550–570°C and 21 kbar for Zone I, 470–500°C and 14–17 kbar for Zone II, 620–670°C and 26–29 kbar for Zone III, 530–560°C and 20–22 kbar for Zone IV, and 490–510°C and 19–20 kbar for Zone V. The symmetrical thermobaric pattern, in conjunction with structural and geochronological data, demonstrates that the Huwan and Hong'an units belong to the same HP slice overlying the UHP slice. This pattern, together with the Mulanshan LT/HP blueschist–greenschist belt in the south, roughly constitutes a ‘normal’ metamorphic zonation. However, clear metamorphic gaps occur between different slices. It is inferred that the LT/HP, HP and UHP slices were broken up from the downgoing slab during subduction and reached different depths along different geothermal gradients. The successive subduction of underlying slices leads to a nearly concomitant uplift of overlying slices, whereas exhumation of the deepest UHP slice was effected by underthrusting of the lower crust of the Yangtze craton.