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Notes: Previous studies have shown that cyclic mechanical stretching exerts anti-inflammatory effects on rabbit chondrocytes. But whether mechanical stretching has similar effects on human tendon fibroblasts are not known. This study therefore aimed to test the hypothesis that cyclic mechanical stretching regulates IL-1β induced COX-2 gene expression in a stretching magnitude-dependent manner. In custom-made silicone dishes, human patellar tendon fibroblasts (HPTFs) were grown on microgrooved culture surfaces, with which the shape and organization of HPTFs in vivo were closely mimicked. To induce inflammatory responses in HPTFs, 10 pM of IL-1β was added to the culture medium. A 4% or 8% cyclic uniaxial stretching was then applied to silicone dishes for 4 hrs using a custom-design stretching apparatus. After the end of stretching, total RNA of stretched and nonstretched tendon fibroblasts was collected, and RT-PCR was performed for measuring COX-2 mRNA expression levels. We found that tendon fibroblasts subjected to 4% stretching decreased COX-2 mRNA expression level by 20%(p = 0.0002; n = 6) compared to that of nonstretched, IL-1β-treated cells. However, cells subjected to 8% stretching showed a 39% increase (p = 0.007, n = 6) in COX-2 mRNA expression. Thus, the results of this study suggest that small stretching (4%) has anti-inflammatory effects on HPTFs in a mildly inflammatory environment such as that induced by 10 pM of IL-1β. In contrast, large stretching (8%) may amplify inflammatory responses of tendon fibroblasts. Therefore, physical therapy with low levels of tendon stretching may be beneficial in reducing mild tendon inflammation.Supported by the Arthritis Investigator Award and NIHAR049921 (JHW)

Notes: Introduction:  As an initial pilot effort a prospective, case-controlled trial examining the effects of treprostinil sodium (Remodulin®), by continuous, subcutaneous infusion, was performed in diabetic patients with recalcitrant lower extremity wounds. While the primary outcomes for the trial were wound healing and limb preservation, evaluation of local tissue perfusion with transcutaneous oximetry and laser Doppler analysis (TcPO2/LD) were included as secondary objectives, and with the expectation they would be useful monitors of any interventional modality.Methods:  All patients enrolled had nonhealing wounds (〉3 months) in critically ischemic lower extremities (Fontaine Stage III-IV or Rutherford Stage 4, 5, or 6). Hyperbaric oxygen exposures were included as diagnostic/monitoring indices. In the last four enrolled subjects TcPO2/LD measurements were made at ambient pressure and at 2.0 or 2.4 ATA before Remodulin® was begun and at various points during drug infusion.Results:  Early, observational evaluation of the TcPO2/LD data shows:1 – These examinations prognostically profile healing in ischemic limbs.2 – TcPO2/LD values demonstrate the efficacy of Remodulin® in augmenting perfusion in ischemic wounds. While there is a broad range, all measured parameters increased on the drug (resting TcPO2 ⇑ 5–30 mmHg, the rate of TcPO2 rise following ambient pressure O2-challenge ⇑ 25–150% and periwound TcPO2 ⇑ 90–300 mmHg at 2.0 and 2.4 ATA).3 – The in-chamber TcPO2 on Remodulin® further defines patients who will not benefit from HBO2 therapy.4 – The TcPO2/LD data will assist in determining dosages, duration, and maintenance requirements for this drug both as a single agent and when used as an adjuvant to HBO2 therapy.Conclusions:  Treprostinil sodium (Remodulin®) has significant laudatory effects on local wound perfusion in ischemic lower extremities.Acknowledgments:  United Therapeutics, Inc provided support for this study through an unrestricted educational grant.

Notes: α1-antichymotrypsin (ACT) and its murine homolog spi2 are serine protease inhibitors present in plasma, that are locally up-regulated during wound healing. Ad-spi2 and Ad-ACT expression vectors were constructed to address the biological activity of locally expressed serpins. The condition medium of infected COS-1 cell at 2 d postinfection contained 1.23 ng/ml spi2 and 2.61 ng/ml ACT. Ad-LacZ or Ad-spi2 108 pfu was injected into the sponges after 3 d subcutaneous implantation in rats. Histological analysis showed that overexpression of spi2 improves granulation tissue organization and collagen deposition at d7 implantation. Moreover, overexpression of spi2 increases the contents of tissue DNA (23.8%, p 〈 0.05) and protein (25.3%, p 〈 0.05) in sponge implantation model in rats. In incisional wound models in both normal and STZ-induced diabetic rats, Ad-spi2 increases tensile strength by 26%(p = 0.011) and 29%(p = 0.022) as well as Ad-ACT increases by 10.3%(p = 0.037) and 32%(p = 0.004), respectively. In adriamycin-induced wound model in rats, histological analysis displayed less inflammatory cells infiltrating in Ad-ACT injected wound than in Ad-LacZ after 21 d injection of adriamycin. Inflammation enzymes MPO and elastase levels were decreased by 32.3% and 29.2% in Ad-ACT injected wound tissue. Our data concluded that overexpression of spi2 and ACT improves wound healing in rats probably via inflammatory resolution.(Supported by Switch Biotech and the Department of Veterans Affairs)

Notes: Cellular interaction with biomaterials determines the fate of the implant. Naturally derived biomaterials such as small intestinal submucosa (SIS) and renal capsule matrix (RCM) may provide optimal structure and composition for cellular repopulation in certain wound healing applications. Porcine renal capsule matrix (RCM) is an acellular, naturally occurring extracellular matrix undergoing characterization and preclinical investigation for the treatment of soft tissue injury. Early studies have demonstrated that RCM constructs induce host tissue infiltration, tissue-specific remodeling, and repair. RCM consists primarily of collagen, although the other proteins have been detected, including the growth factors basic FGF, VEGF, and CTGF.Transmission and scanning electron microscopy were used to envision the cellular environment provided by this biomaterial. Comparison of the images utilizing cryo and traditional preparation techniques show that RCM is a dense, heterogeneous matrix consisting of textured fibers of mixed size. Although the matrix architecture shows signs of collapse following lyophilization, much of the native structure seen in the hydrated isolate is preserved. These differences are maintained following rehydration in normal saline. The effects of these structural changes on cellular reaction and infiltration are unknown, but the naturally complex architecture likely facilitates host interaction at the cellular level. Further compositional characterization is under way.This work was supported by Cook Biotech Incorporated, West Lafayette, IN.

Notes: The use of fresh platelets has gained value in medicine as an essential part of wound treatments. This is not surprising since platelets contain a number of bioactive factors that contribute to the process of wound healing, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF). Fresh platelets’ short shelf life limits platelet-based therapies. If platelets can be stabilized in freeze-dried form (FDP) then long-term storage as well as pathogen inactivation methods become possible. Adlyfe and Oregon Freeze-Dry have been developing technology to stabilize freeze-dried human platelets that can be subjected to gamma irradiation and stored for a long duration. Upon reconstitution, irradiated FDP retained growth factors PDGF-AB, PDGF-BB, and TGF-B1 in quantities similar to fresh platelets as judged by capture ELISA. The rehydrated FDP promoted new DNA synthesis and cellular proliferation of primary human dermal fibroblasts and endothelial cells (HUVECs) similar to fresh platelets. The FDP also promoted remodeling of extracellular matrix by accelerating fibroblast-mediated contraction of collagen gels and stimulated HUVECs to undergo angiogenesis and form capillary structures in vitro. Therefore, we conclude that FDP and fresh platelets have comparable in vitro wound healing potential. Preclinical wound healing stud"ies in diabetic mice are under way and further development will allow FDP to be a safe and well-suited alternative to fresh platelets for wound healing applications.

Notes: To investigate changes in the mechanical properties of skin with injury, tissue was evaluated 70 days following full thickness wounding in juvenile female Yorkshire pigs (N = 14). Samples were taken in the axial (cranial-caudal) and transverse (dorsal-ventral) directions, for both scar tissue and uninjured skin. The samples were evaluated mechanically in vitro using a protocol of stress relaxation followed by tensile failure, in an INSTRON universal test machine. Specimens were subjected to a 10% elongation and held for a 4-min isometric period, after which they were returned to the original length and failed in tension.Axial normal skin exhibited a smaller force at 10% elongation (−58%, p = 0.002), and higher failure load (p = 0.027) and displacement (p = 0.028) than transverse. Scarring reduced the failure strength on average by 60% in the axial and 65% in the transverse directions (p 〈 0.001), with large decreases (〉50%) in displacement at failure in both directions (p 〈 0.001). Elongation of 10% produced a larger force in axial scars (p 〈 0.001) than their normal counterparts, indicating a reduction in small-displacement compliance. Axial scars exhibited greater failure strength (p = 0.009) than transverse scars, yet no significant differences were observed in elongation to failure or load at 10% elongation.These results indicate that normal skin exhibits higher compliance and failure strength in the axial direction. Following injury, the strength and compliance of the tissue are greatly reduced, and the strong compliance directionality originally observed, is lost. Therefore it appears that the healing response leads to increases in tissue stiffness, such that small displacements result in larger tissue loads. This is further complicated by the reduced ability of the scar to resist failure. Taken together, these findings illustrate the compounding negative effects of scar on tissue integrity and function.Acknowledgments:  Alberta Ingenuity Fund, CIHR and NSERC

Notes: Fibroblasts from a healing ligament likely have differential phenotypical expression from those in an intact ligament, because fibroblasts at the healing site need to repair/regenerate the wounded matrix around them caused by injury, whereas normal fibroblasts just maintain an intact matrix. This study was designed to determine the differences in proliferation, collagen production, α-smooth muscle actin (α-SMA) expression, and contraction between healing and normal fibroblasts. Transected and sham-operated rat medial collateral ligaments (MCL) were used to obtain healing and normal fibroblasts, respectively. It was noted that healing and normal MCL fibroblasts in culture were seen to have a similar morphology. However, healing fibroblasts in monolayer culture proliferated 1.4-fold faster at 48 hours and had 1.7-fold greater protein expression of α-SMA than normal fibroblasts. In addition, it was noted that the proliferation of healing fibroblasts in collagen gels was not significantly different from that of normal fibroblasts at 24 hours, but it was at 48 hours. Furthermore, in collagen gels, healing fibroblasts produced 10% more type I collagen than normal fibroblasts and generated 1.6- and 1.7-fold larger contractile forces at 15 and 20 hours, respectively, than their normal counterparts. Taken together, the results of this study show that healing fibroblasts possess a differential proliferation, α-SMA protein expression, and contraction than normal fibroblasts. This study also highlights the importance of using healing fibroblasts to study the cellular and molecular mechanisms of ligament healing.Supported by the NIH grant AR049921 and Whitaker Foundation Grant (JHW)

Notes: Cultured skin substitutes (CSS) are used as an adjunctive treatment for closure of massive burn wounds. CSS are prepared with keratinocytes and fibroblasts combined with a collagen-based matrix. A cDNA microarray analysis was performed to characterize CSS at a molecular level. We hypothesized that combination of keratinocytes and fibroblasts in a three-dimensional organo typic culture system would result in changes in gene expression compared with cells grown in monolayer culture. Human skin samples were obtained from breast and abdominal tissue of healthy adult females. Fibroblasts and kera tinocytes were isolated and grown in monolayer cultures; CSS, prepared with cells from four independent donors, were cultured for 2 weeks in vitro. Microarray analysis was performed on RNA from fibroblasts, keratinocytes, and CSS using the Affymetrix Human Genome U133A gene chip representing over 22,000 human genes. Cluster analysis was performed to identify gene expression “signatures” for each group, and further analysis was performed to identify genes that were significantly increased or decreased in CSS compared with either cell type. Microarray analysis revealed significant changes in gene expression upon combination of fibroblasts and keratinocytes in organotypic culture. Genes belonging to several classes were significantly increased in CSS compared with fibroblasts and keratinocytes. For example, genes associated with epidermal differentiation, including fillagrin, corneodesmosin, involucrin, and multiple keratins (1, 10, 13, 16, 23), were increased in CSS. Several genes that are overexpressed in psoriasis (S100A7, SERPINB3, PSORS1C1), as well as cytokines IL- 6 and IL-8, were also increased in CSS. Further, multiple MMP genes were up-regulated in CSS. The results suggest that epithelial-mesenchymal interactions contribute to morphogenesis of CSS, including the remodeling of the dermal layer and stratification and differentiation of the epidermal layer. This analysis reveals that CSS exhibit many characteristics of normal or inflamed human skin.

Notes: There are only a few models for studying epidermal migration in vitro, and their interpretation is made difficult by reliance on cell monolayers and the choice of a specific substrate. In this study, the process of keratinocyte migration and epiboly were investigated by using a bilayered bioengineered skin construct, consisting of human neonatal foreskin keratinocytes and dermal fibroblasts (Apligraf, Organogenesis, Canton, MA). At baseline, 6-mm punch biopsies of the construct were placed in serum-free media (AIM-V) or DMEM with or without 10% FBS. At varying time points the bioengineered skin samples were processed and analyzed by histology and immunostaining. By 72 hours, in a time-dependent manner, the epidermis had migrated over and enveloped the entire dermis (full epiboly). Full epiboly was partially inhibited by serum and was maximal in serum-free medium. Epiboly was preserved 5 days after stated expiration date and was equivalent to that seen in unexpired construct when stored at room temperature. The process of epiboly was downregulated in a dose-dependent manner by neutralizing antibodies to EGF and TGF-beta 1. The migrating epithelium was characterized by decreased keratinocyte proliferation (as per Ki67 immunostaining) and increased expression of vitronectin (epibolin). Increasing concentrations of antibodies of vitronectin blocked the process of epiboly, as did antibodies to the alpha5-betaV integrin receptor, which mediates vitronectin-driven keratinocyte locomotion. Interesting, epiboly was also blocked by preseeding human dermal fibroblasts on the dermal side of the construct. We propose that the process of epiboly in this model can be used to better understand and assess the mechanisms involved in keratinocyte migration and may be used as an assay for establishing construct functional viability.

Notes: We find that the ELR-negative CXC chemokines, CXCL10 (IP-10) and CXCL11(IP-9), expressed during wound repair, with IP-9 being produced by redifferentiated keratinocyte. Immunohistochemical analysis for IP-9 on human full thickness wound tissues collected from day 5 confirmed that IP-9 is a wound response protein. We have shown in in vitro coculture systems that IP-9 can serve as an autocrine factor promoting motility in keratinocyte while limiting motility in dermal fibroblasts. These effects occur via modulation of the intracellular proteases calpain isoforms that regulate cell adhesion during motility. Thus, the function of these chemokines in vivo is not obvious. To probe this, we have explored wound repair in mice lacking the receptor for these ligands, CXCR3. Quantitative analysis of IP-9 expression in wounds of these mice collected from day 2 to day 20 showed a significant increase in IP-9 levels in CXCR3-/- mice compared to wild type, which is expected if receptor-mediated feedback attenuation is lost. Full and partial thickness wound healing experiments on these mice showed a significant delay in CXCR3-/- mice when compared to wild type. Histochemical analysis of tissues collected from both full and partial thickness wounds demonstrated differences in epithelialization, granulation tissue formation, and angiogenesis in CXCR3-/- mice when compared to wild type. Collagen, the major extracellular matrix protein organization were analyzed by trichrome and picrosirus red staining methods. We are also examining the effect of this lost receptor on promotility signaling from EGF receptors. These in vivo and in vitro studies confirm the pathophysiologic role of the chemokines IP-9 and IP-10 and their cognate receptor CXCR3 during wound repair.

Notes: In this study, we evaluated and compared the effectivity of two modalities, gene therapy with adenovirus-mediated transforming growth factor-β and extracorporal shock waves (ESW) therapy, to treat ischemically challenged epigastric skin flaps in a rat model. Thirty male Sprague-Dawley rats were divided into three groups of ten rats each. An epigastric skin flap model, based solely on the right inferior epigastric vessels, was used as the model in this study. Rats received either subdermal injections of adenovirus encoding TGF-β(Ad-TGF-β) or ESW treatment with 750 impulses at 0.15 mJ/mm2. The third group received no treatment and served as a control group. A flap measuring 8 × 8 cm was outlined on the abdominal skin. The TGF-β injections were given subdermally in the left upper corner of the flap just prior to flap elevation, whereas the ESW treatment was immediately after the flap was sutured back to its bed. Flap viability was evaluated seven days after the initial operation. Digital images of the epigastric flaps were taken and areas of necrotic zones relative to total flap surface area were measured and expressed as percentages by using a software program. Overall, there was a significant increase in mean percent surviving area in the Ad-TGF-β group and the ESW-group compared to the control group (ESW-group: 97.7 ± 1.8% vs. Ad-TGF-β: 90.3 ± 4.0% and control group: 82.6 ± 4.3%; p 〈 0.05). Furthermore, in the ESW group mean percent surviving areas were significantly larger than in the Ad-TGF-β group (ESW-group: 97.7 ± 1.8% vs. Ad-TGF-β: 90.3 ± 4.0%; p 〈 0.05).We conclude that treatment with ESW enhances epigastric skin flap survival significantly more than Ad-TGF-β treatment and, thus, represents a modality that is more feasible, cost effective and less invasive compared to gene therapy with growth factors to improve blood supply to ischemic tissue.

Notes: Homeobox genes are transcription factor that form nested patterns in specific regions throughout the body. Homeobox genes are important for wound healing study because some have been shown to be related to scarless fetal wound healing. Muscle segment homeobox-2 (Msx-2) is a homeobox gene commonly expressed at sites of epithelial-mesenchymal interactions during tissue morphogenesis. Overexpression of Msx-2 leads to craniosynostosis, epidermal dysplasia, and abnormal hair growth. A knockout of this gene causes pleiotropic defects in bone growth, cyclic alopecia, and a thinner dermis. While Msx-2 has been found to play many critical roles in development, no functional study has been conducted on Msx-2 and wound healing. We hypothesize that Msx-2 regulates skin morphogenesis during wound repair which was tested by studying excisional skin wound closure. Isolated keratinocytes and dermal fibroblasts of Msx-2 WT and KO mice were also studied to determine the effects of Msx-2. Results showed that Msx-2 mRNA was induced in epidermis and dermis during wound repair. Additionally, Msx-2 KO mice exhibited enhanced re-epithelialization and faster wound closure than the WT control. These repair phenotypes may be attributed to keratinocyte motility and fibroblast interaction with collagen matrix as, in vitro, Msx-2 KO keratinocytes exhibited increased motility, and fibroblasts show stronger collagen matrix contraction and expressed higher levels of α2β1 than WT control. Msx-2 KO fibroblasts were refractory to BMP4 in collagen matrix contraction, demonstrating a disruption in the Msx-2 pathway. In conclusion, Msx-2 may regulate skin morphogenesis during repair at both epithelial and mesenchymal levels.This work was supported by NIH grant R01GM055081(TLT).

Notes: Percutaneous devices play a central role in modern medicine, but are associated with significant risk of infection. One approach to circumvent this risk is to heal the wound around the device by creating a seal at the skin/device interface. We previously showed, using cell adhesion assay and organ culture model, that surface modification of poly(2-hydroxyethyl methacrylate)[poly(HEMA)] with 1,1′-carbonyldiimidazole (CDI) dramatically enhanced keratinocyte attachment to poly(HEMA). Although the organ culture model addresses epithelial response to an implanted biomaterial, to fully evaluate the cutaneous interaction with biomaterials, we tested implants in a mouse model. Porous poly(HEMA) rods with 40 μm pore size were implanted into two different sites on dorsal skin of forty eight, 6–8 week old male C57BL/10 J mice. Porous poly(HEMA) rods were treated with 1) PBS, 2) CDI, and 3) CDI plus laminin 5. Samples were harvested at 7, 14, and 28 days after surgery and analyzed morphologically using H&E and immunohistochemistry. Implanted rods remained intact and infection free at all time points. Morphological analysis showed diffuse dermal cellular infiltration in all samples and evidence of epidermal integration into the pores of CDI treated poly(HEMA) and CDI plus laminin 5 treated poly(HEMA). Immunohistochemistry showed laminin 5 expression at the skin/poly(HEMA) interface resembling the junctional epithelium of the tooth. Engineering this type of attachment to percutaneous devices is attractive since the tooth is a natural example of percutaneous material, where bacterial invasion of bone is prevented by epithelial attachment.Acknowledgments:  NSF EEC 9529161 [University of Washington Engineered Biomaterials (UWEB)], NIH DK 59221, the George F. Odland Endowed Research Fund, and the Marvin and Judy Young Research Fund

Notes: Introduction:  Clinically, it has been observed that treatment with an occlusive dressing that results in hydration of the epidermis reduces scarring. A keratinocyte/fibroblast coculture system in which the epidermis is hydrated results in increased TNF production by keratinocytes, and reduced collagen synthesis by fibroblasts. We wished to test the hypothesis that in a nonhydrated coculture system, increasing TNF expression would result in decreased collagen synthesis by fibroblasts. Both keratinocytes and fibroblasts play essential role in scar formation. However, little is known about how TNF-alpha specific communication between these cell types modulates scar formation. To better elucidate this interaction, we used a keratinocyte/fibroblast coculture system to assess fibroblast function in a milieu of TNF-alpha overexpression by keratinocytes.Methods:  Primary human keratinocytes were transfected with adenovirus (MOI = 10) containing cDNA for TNF-alpha (AdTNF-alpha) or with LacZ and then cocultured with human dermal fibroblasts for 6 days.Results:  TNF-alpha transfected keratinocytes and their corresponding conditioned media showed a 3000-fold increase of TNF-alpha expression and synthesis in QRT-PCR and ELISA. Subsequent antibody blocking experiments with a specific TNF-alpha antibody showed a dose-dependent increase in collagen type 1 protein in the conditioned media. Col-1A1 mRNA expression in dermal fibroblasts was significantly reduced (p 〈 0.05).Conclusion:  Our data demonstrate that in a coculture setting, TNF-alpha expression by keratinocytes reduces Col-1A1 expression in fibroblasts and suggests a mechanism for clinical finding on the scar-reducing effects of hydration. This may bolster support for a TNF-alpha-mediated therapeutic intervention to ameliorate wound scarification.

Notes: A delicate balance between synthesis of extracellular matrix (ECM) and degradation by matrix metalloproteinases (MMPs) is a key factor in maintaining the structural integrity of normal skin. Epidermal-mesenchymal interactions play a critical role in controlling the expression of MMPs during development and healing of skin. Disruption of this interaction increases the frequency of developing fibrotic conditions such as hypertrophic scarring and keloids. In the absence of epithelialization, ECM continues to accumulate until dermal fibroblasts receive signal(s) from epidermal cells to slow down the dynamic process of maturation and remodeling of the healing wound. We have recently demonstrated that keratinocyte releasable stratifin stimulates MMP-1 expression in dermal fibroblasts. However, the molecular mechanism by which stratifin protein induces MMP-1 expression in fibroblasts is unknown. Therefore, the purpose of the present study is to identify elements of the signaling pathway mediating stratifin stimulation of fibroblast MMP-1 expression. We did so by examining the three distinct MAPK pathways: ERK1/2, JNK, and p38 as well as the expression of the main components of the AP-1 dimers, c-Jun and c-Fos, which are mediated by distinct MAPK pathways. Our data shows that treatment of fibroblasts with stratifin resulted in rapid and transient up-regulation of c-jun and c-fos mRNA levels. Furthermore, we show that stratifin activates fibroblast MMP-1 expression at the mRNA and protein levels and this is mediated by p38 MAP kinase. Subsequent cDNA microarray analysis of fibroblasts treated with stratifin show an increase in a ternary complex factor, Sap-1. In conclusion, our results describes the mechanism by which stratifin activates MMP-1 in fibroblast, and demonstrates that p38 MAP kinase is an important regulator of MMP-1 gene expression involved in epidermal-mesenchymal interactions in wound remodeling.Acknowledgment:  This work was supported by the Canadian Institute of Health Research (CIHR).

Notes: The Hypoxia Inducible Factor (HIF) system has been characterized as the principal tissue level response to hypoxia. Posttranslational regulation of HIF-1alpha has been reported to act though a Hypoxia Responsive Element (HRE) promoter region on a range of hypoxia-induced genes. A plasmid was constructed consisting of a fivefold HRE repeat conjugated to a luciferase gene, used as a marker for HRE activation. Plasmid HRE-luciferase was then transfected into a well-established ischemic rabbit ear wound healing model. Ischemia was induced using a variety of published models for interruption of arterial inflow and compared with a nonischemic control. Central artery, caudal artery and rostrocaudal artery models were tested for induction of ischemia. Tissue specimens were harvested from the transfected areas and solubilized. Luminescence of each specimen was measured using a standard luminometer to quantify luciferase induction. To provide correlation on a regional level, tissue level oxygen tension was measured directly for each wound model. Profound and statistically significant differences were found in the induced luciferase production. Marked differences in the tissue hypoxia between the various ischemic wound healing models correlated in graded fashion with the ischemia induced on an anatomic basis. The least ischemic model showed no significant difference in hypoxia readings versus control. The intermediate model showed a significant fourfold greater ischemia signaling. The most ischemic model showed a highly significant 72-fold greater ischemic response compared to controls. The use of gene transfection is described as a sensitive and effective method for quantification of tissue hypoxia at a cellular level in ischemic wounds.Acknowledgments:  This study was funded by the Wound Healing Research Laboratory.

Notes: Impaired wound healing is a problem for immobilized patients, diabetics, and the elderly. The 43 amino acid angiogenic peptide thymosin β4 has previously been found to promote accelerated dermal wound repair in rats, aged mice and db/db diabetic mice, and corneal repair in normal rats. It has been found in great abundance in wound fluid. Here, we hypothesized that thymosin β4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Western blot analysis of keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of thymosin β4 showed changes in the expression of the MMP-1, −2, and −9. Zymographic analysis of whole excised mouse wounds taken after homogenization also showed changes in MMP-2 and-9 expression over a 3-day period. These results were confirmed in 2-day-old wounds by RT-PCR. We conclude that part of the wound healing activity of thymosin β4 resides in its ability to increase protease activity. Since thymosin β4-induced protease activity can be further controlled by inflammatory cytokines, a regulatory role for thymosin β4 is proposed in wound healing. These studies suggest that thymosin β4 may play a pivotal role in extracellular matrix remodeling during wound repair and may be effective in the treatment of chronic wounds in humans.

Notes: The large bulk of lost craniofacial soft tissue is mainly adipose tissue. Current surgical approaches following tumor resection and trauma have limitations. Adipose derived-stromal cells (ADSCs), also named as preadipocytes, have been demonstrated to be a good cell candidate for adipose tissue engineering. However, characteristics of scaffold played important role in a successful tissue engineered adipose tissue.Objectives:  The present study is to investigate whether in vitro adipogenesis could be induced both in a monolayer and 3-D by ADSCs.Methods:  Healthy adult ADSCs were isolated by liposuction from fat pads. After discarding the unattached cells, the ADSCs were cultured in DMEM supplemented with 10% FBS and 1% antibiotics until they reached confluence. After trypsinization, ADSCs were subcultured at a density of 105 cells/100 mm-dish as a first passage. The monolayer and 3-D adipogenesis were assessed after seeding 105 ADSCs into each well of 6-well plates and 5 × 106 ADSCs/ml into biodegradable collagen sponges, respectively, followed by replacement of basic medium with adipogenic one.Results:  In both monolayer and 3-D scaffolds, lipids containing adipocytes were observed 1 week after adipogenic stimulation, and further increased over the time. By contrast, no adipogenesis was observed in ADSCs cultured in basic medium.Conclusions:  Human adipose-derived stromal cells isolated from fat pads can readily be induced toward adipogenesis in vitro. The in vivo adipogenesis potential of ADSCs is being investigated.(Supported by Whitaker Biomedical Engineering Foundation, March of Dimes Foundation)

Notes: Background:  Apligraf, a bioengineered living skin construct, has been shown to accelerate the healing of venous leg and diabetic foot ulcerations. It appears to function as cell-based therapy (e.g., delivering growth factors), rather than as a true graft. Given this mechanism of action, its use could be expanded to areas in which traditional skin grafting is not routinely employed.Methods:  A 64-year-old wheel-chairbound Caucasian male presented to the wound clinic with a grade III left trochanteric pressure ulcer which had been present since 1976. He had been seen by numerous physicians and treated with debridement, topical antimicrobials, negative pressure therapy, growth factors, and a variety of dressings. We spent several months preparing the wound bed: maintaining adequate moisture balance, performing serial debridement, utilizing offloading surfaces and reducing the bacterial burden. His nutritional status was optimized. The wound bed developed a healthy granulating base but failed to close.Results:  A single unit of Apligraf was meshed at a 1:1.5 ratio and applied to the wound. It was fixed in place using a nonstick silicone dressing, foam, and a self-adhesive covering. The dressing was changed weekly in the wound clinic. The initial response was a decrease in wound depth followed by steady epithelialization. Complete closure occurred 17 weeks after grafting. The wound has not recurred.Conclusion:  This single case study suggests that bilayered cell therapy may have application in difficult wounds, such as pressure ulcerations. Further study into the efficacy of Apligraf in pressure ulcerations is ongoing.

Notes: Various matrices containing both synthetic or natural polymers are currently used for local regional drug delivery to a number of disease locations such as chronic cutaneous wounds, joints, bone, muscle, and nerves. Insert Therapeutics has developed a novel synthetic matrix which consists of a copolymer of β-cyclodextrin and polyethylene-glycol (PEG). Addition of di- or multifunctionalized adamantane-PEG molecules results in noncovalent cross-linking through inclusion complex formation. The resulting hydrogel showed rheological characteristics amenable to topical application or local-regional injection into a variety of tissues (Bellocq et al. (2004) Bioconjug Chem 15(6): 1201–1211).This matrix was shown to be biocompatible, allowing for cellular growth and migration in vitro. It also allowed for efficient delivery of an adenovirus gene therapy vector to dermal fibroblast cells. In vivo, the matrix demonstrated efficient delivery of biotherapeutics such as recombinant adenovirus and non-viral gene therapy vectors after intradermal injection. The matrix was well tolerated and was as efficient as collagen in promoting wound healing when an adenoviral gene therapy vector expressing PDGF-bb was delivered to cutaneous wounds of diabetic mice.The matrix can additionally be modified to incorporate therapeutic small molecules, peptides, or proteins, either through inclusion complex formation or chemical linkage. Using biodegradable linker chemistry, the release rate of covalently attached molecules can be controlled. The cyclodextrin-PEG matrix is therefore an attractive alternative to existing matrices that is biocompatible, biodegradable, tunable with regard to its physicochemical properties, and can be designed to deliver multiple therapeutic agents to a variety of tissues in a controlled fashion.

Notes: Neuropathic ulcers represent a significant cause of morbitity for diabetics in the United States. Diabetic wounds are notoriously difficult to resolve. Treatment options are limited due in part to the lack of knowledge about the molecular events that regulate successful resolution of wounds. To identify differences between normal and diabetic gene regulation in wounds, we examined the level of expression of 12,000 genes in granulation tissue and wound-edge epithelium in normal animals and in an experimental model of diabetes following induction of full thickness skin injuries. Full thickness injuries in diabetic mice were characterized in part by altered regulation of structural genes and genes that regulate energy metabolism and utilization. The energy cycle contributes backbone structures for the synthesis of a variety of amino acids, the molecular building blocks essential for wound repair. A direct consequence of a diminished ability to efficiently exploit energy supplies would be decreased availability of regulatory and structural proteins necessary for efficient healing in diabetic wounds. We have successfully enhanced healing in diabetic wounds by treatment with the angiotensin II peptide NorLeu3A(1–7). NorLeu3A(1–7) accelerates healing of full thickness injuries in diabetic mice. While NorLeu3A(1–7) enhances healing by accelerating collagen deposition and reepithelialization, gene array studies show that NorLeu3A(1–7) acts locally within the wound site by altering gene expression within the granulation tissue. Specifically at day 7 after injury, NorLeu3A(1–7) reduced the expression of MMP genes and increase the expression of genes involved in energy metabolism.Supported by PHS grant NIAMS 2 R44 AR47481

Notes: Antimicrobial peptides are major components of the innate defense system that present microbicidal activity against gram positive and negative bacteria, yeast, and some enveloped viruses. The human cathelicidin LL-37 is expressed in skin upon wounding and, in addition to its role in antimicrobial defense, it has also been previously demonstrated that this peptide might be involved in reepithelization. We have generated an adenoviral vector containing the complete sequence of LL-37 along with an Ires-GFP expression cassette. The efficacy of this vector was probed by testing the conditioned medium from adenoviral transduced keratinocytes in preventing bacterial growth. We have studied the in vitro effects of LL-37 on HK migration and proliferation. In addition, we have efficiently transduced human skin grafts (in immunodeficient mice) using an in vivo gene transfer approach and we are currently testing the involvement of this peptide in wound repair.

Notes: Fluorescence correlation spectroscopy was used to measure the binding and diffusion of growth factors in model extracellular matrices in order to investigate the importance of protein-matrix interactions in regulating signaling molecules within a three-dimensional matrix. Two important growth factors were studied, transforming growth factor β1 and basic fibroblast growth factor, which are known to have specific affinities for fibronectin and the heparan-sulfate-proteoglycan perlecan, respectively. Collagen-based matrices were prepared by polymerizing type I collagen in the presence of fibronectin or perlecan, and we measured diffusion constants and binding constants of the two growth factors. The binding constant measured for transforming growth factor β1 with fibronectin-containing matrices was in good agreement with that measured using affinity chromatography. However, the interactions measured between fibroblast growth factor and perlecan were significantly weaker than expected. Surprisingly, the strongest interactions by far were with monomeric collagen solutions and fibrillar collagen matrices. Our findings suggest a central role for the three-dimensional fibrillar collagen matrix in growth factor interactions, with modulatory roles for fibronectin or perlecan.

Notes: Platelet-derived growth factor (PDGF-B) is a potent cell mitogen and chemotactic signal involved in normal wound healing. PDGF-B is present at decreased levels in diabetic wounds and has been shown to improve wound healing when applied in a topical form. However, clinical results with topical PDGF in the treatment of diabetic wounds have been equivocal. In this study, we investigated whether application of PDGF-B via gene therapy can effectively promote wound healing in the diabetic environment in an animal model. Retroviral vectors carrying the gene for human PDGF-B were constructed using the LNCX virus and the G418 resistance gene for selection (LN-PDGF-B), and transduced into mouse dermal fibroblasts. LN-PDGF-B transduced fibroblasts were seeded into alginate hydrogel scaffolds at a concentration of 25 × 106 cells/implant and implanted into 2 cm × 2 cm full thickness excisional dermal wounds created on the dorsae of genetically diabetic db/db mice. At 21 days, animals treated with LN-PDGF-B transduced cells suspended in alginate hydrogel demonstrated a significantly smaller epithelial gap (0.97 +/− 0.34 cm) than animals receiving untransduced mouse dermal fibroblasts alone (1.44 +/− 0.37 cm; P 〈 0.05) or animals receiving dermal fibroblasts transduced with LNCX viral vector alone (1.40 +/− 0.20 cm; P 〈 0.01). These results suggest tissue-engineered retroviral gene therapy with PDGF-B specifically promotes diabetic wound healing and may yield advances in the effective treatment of diabetic wounds.Supported by a grant from the American Diabetes Association

Notes: Nitric oxide (NO) is a critical mediator of normal tissue repair and is inhibited by homocysteine (Hcy). Following 8 weeks of lower extremity ulcer (LEU) treatment with human fibroblast-derived dermal substitute (Dermagraft®) for 12 (n = 12) patients, a correlation was observed between patients with a poor wound healing response to Dermagraft and elevated serum homocysteine (Hcy). The responders group (R) to Dermagraft treatment (n = 6) was observed with robust granulation tissue, epidermal migration and a 67% rate of healed ulcerations. All R-group patients (6/6) had normal serum Hcy. The nonresponders (NR) group (n = 6) exhibited poor granulation tissue formation and epidermal migration and a complete absence of healed wounds. Five of the NR patients (5/6) were observed with elevated serum Hcy. There was no significant (p 〈 0.05) difference between the wound areas (cm2 ± SE) of each group [35.43(±28.99)-R vs. 19.90(±7.55)-NR]. After 2 weeks of treatment, the R-group demonstrated significantly greater %Δwound area than the NR-group [62.17 (±7.96)-R group vs. 23.17(±8.82)-NR group; p 〈 0.05]. Wound fluid nitrate (WFNOx), a surrogate measurement (μM ± SE) for local NO bioactivity, was significantly lower for the NR-group than the R-group [3.17(±1.46)μM-NR group vs. 12.98(±1.73)μM-R group; p 〈 0.05]. In a large group of LEU patients (n = 138) we observed a 50% incidence of elevated Hcy; 69% for diabetic neuropathic LEU patients and 47% for nondiabetic LEU patients. Hcy inhibits NO production by multiple pathways and may also inhibit wound repair by occupying the fibronectin domain of fibrin during provisional matrix formation. Our study suggests that elevated serum Hcy is a common finding in chronic LEU patients. The high Hcy may contribute to impaired wound healing by decreasing wound NO production and, perhaps, by inhibition of fibronectin-fibrin-mediated wound matrix development.

Notes: Atherosclerotic plaque formation involves recruitment and adhesion of circulating monocytes to sites of blood vessel wall injury followed by their migration into the subendothelial space stimulated by MCP-1 (monocyte-chemoattractant-protein1). Monocytes differentiate into macrophages, cells that release a variety of factors and take up oxidized LDL, becoming foam cells. Smooth muscle cells of the vessel wall differentiate and migrate to the subendothelial space, interact with foam cells and components of the ECM to form a “fatty streak,” the precursor of plaque. Adiponectin, a protein secreted by adipocytes, circulates in the blood, maintains blood vessel wall stability and inhibits foam cell formation. Cigarette smoke is a major risk factor of atherosclerosis, although the mechanisms remain unknown. We showed previously that both firsthand and secondhand cigarette smoke stimulate MCP-1 in endothelial cells and others have shown that adiponectin levels in smokers with cardiovascular disease are reduced. We hypothesized that a localized increase in MCP-1 and a decrease in adiponectin are critical for cigarette smoke-induced plaque formation. To test this possibility, we used mice transgenic for human ApoB100, a model system that closely mimics human conditions that lead to atherosclerosis, and a smoking machine that closely simulates human smoking. We found that, in smoking mice, the micro vessels in the heart tissue are often filled with lipids, the areas surrounding the plaque-prone regions have numerous neutrophils, that these cells express high levels of MCP-1, and that the plaques are larger than in control mice. In addition, the level of the adiponectin monomer in the blood of smoking mice is lower and the presence of this protein in the heart tissue is also decreased. In conclusion, the decrease in adiponectin and the increase in MCP-1 levels may be responsible for cigarette smoke-induced atherogenesis.

Notes: The treatment of venous ulcers must start with compression and if the ankle brachial index is greater than 0.8 high compression bandages can be applied. Despite edema control, there are a number of venous ulcers that do not heal at the expected rate. Patients with venous ulcers of greater than 4 weeks duration were treated with a prolonged release absorptive nanocrystalline silver dressing (Acticoat 7) under the 4 layer bandage, Profore for 12 weeks, or until healing. Biopsies were obtained from the ulcer base at week 0 for histology and bacterial burden. Duplicate biopsies for quantitative bacteriology were performed with one submitted whole and the second bisected into superficial and deep components. The paired biopsies were then repeated after a median of 6.5 weeks (range 2 to 12 weeks). The histological specimens were examined by the histopathologist (SR). Inflammatory infiltrates were identified in the superficial, middle and deep segments of the biopsies. Acute infiltrates were identified through the concentration of neutrophils and chronic infiltrates by the presence of lymphocytes. Each biopsy and each segment was graded for infiltrates on a four point semi quantitative score. A total of 15 patients (9 male, and 6 female) were enrolled into the study. The median age was 63 years (range 30–83 years). The median duration of current ulceration was 17.3 weeks (range 4 weeks to 11 years) and the median ulcer area was 4.8 cm2(range 1.8–43.9 cm2). The median exposure to Acticoat 7 was 82 days (range 8–86 days).There were 12 sets of paired biopsies that were analyzed. There was a statistically significant reduction (p = 0.0114) in the log10(total bacterial count) between the baseline and final biopsies (median 4.48 and 3.00, respectively). Four patients healed, 8 patients continued to the end of the 12-week study period and three patients were discontinued early. For all patients, the median percentage reduction in ulcer area was 94.4% and the median final ulcer area was 0.4 cm2. Analysis of the histology and bacteriology data demonstrated that the presence of a high neutrophilic infiltrate in skin biopsies was associated with high bacterial counts (superficial compartment of the quantitative biopsies) at week 4 and delayed healing (p = 0.037). In the week 0 biopsy, increased lymphocytic infiltrates within the superficial and middle segments were associated with accelerated healing in the first 4 weeks (p = 0.26 and 0.09). The nanocrystalline silver dressing has demonstrated an anti-bacterial and permissive but selective anti-inflammatory action allowing lymphocytic infiltrates to increase associated with an accelerated reduction in ulcer size.

Notes: Advocate Christ hospital is a 650-bed, level-one tertiary-care center that created a comprehensive wound care program in November 1998. Over the first 3 years of the program there were over 140 admissions per year directly from the wound center along with numerous hospital to hospital transfers for complex wound management. Prevalence rates therefore increased linearly over this time frame. The wound team calculated a system-wide (eight hospitals) spend of $2.1 million per year for specialty rental beds. Incidence rates for the system were at, but not lower than, national levels despite the high cost incurred. A $1.3 million capital purchase for KCI atmos-air 9000/〉 mattresses resulted in the complete replacement of all medical surgical beds in all eight hospitals. An internal prevalence and incidence team was created and data were collected both at the hospital and system-wide level on a quarterly basis. Hospital and system incidence rates fell to or below 7%(targeted goal) and the entire project will achieve a return on investment in 18 months. Data to be presented include prevalence and incidence results, rental bed costs pre- and postbed purchase, and a detailed review of the process of creating an electronic prevalence and incidence tracking system.Acknowledgments:  This study was not supported by any outside grants.

Notes: We have previously shown that resident fibroblasts from human chronic wounds are phenotypically abnormal, displaying an impaired response to TGF-beta 1, decreased TGF-beta Type II receptor expression, and decreased phosphorylation of Smad2, Smad3, and p42/44 MAPK. We now report that these human cells delay healing by approximately 30% when implanted in mouse incisional wounds and in a dorsal flap model. These new findings point to the need for the transplantation of new healthy cells in human chronic wounds. Bioengineered living skin constructs can deliver new cells to chronic wounds, at least for a limited period of time. However, most available bioengineered skin constructs consist of already differentiated allogeneic cells, which do not allow for the possibility of engraftment and reconstitution of wound structures. Recently, in a preliminary report, we have shown that autologous bone marrow-derived cells can dramatically enhance the healing of recalcitrant chronic wounds when all other treatments have failed. Our present efforts are now focused on characterizing the bone marrow cells involved in this stimulation of wound healing and in developing better ways for their topical delivery to the wound. Here we present evidence that a fibrin construct may be an ideal way of applying bone marrow-derived cells to human chronic wounds. Autologous bone marrow cells are allowed to grow in culture under conditions favoring the survival and proliferation of mesenchymal stem cells (MSC), as shown by surface markers and functional studies. This usually requires two weeks after bone marrow harvesting. The cellular component is then placed in a fibrinogen solution which, when simultaneously combined with thrombin through a CO2 driven flow, delivers a fine cell-containing fibrin polymer film to the wound. In vitro and in vivo experiments, both in mouse models and in human wounds, show the feasibility of this approach.

Notes: Bone marrow stromal cells (MSCs) are a promising cell resource of osteoprogenitor cells for bone tissue engineering. However, the diverse characteristics of osteoprogenitor cells within the bone marrow of individual subjects require varying treatments to stimulate osteogenic differentiation. Thus, an effective monitoring system is needed to identify the progression of osteogenesis. Magnetic resonance (MR) microscopy was used in the present study to monitor osteogenesis of tissue engineering (TE) constructs prepared by human bone MSCs seeded on scaffolds of gelatin sponges. The characteristics of MR images and parameters corresponded to osteogenic progression of TE constructs exposed to differentiation medium, significantly differing from control groups exposed to basic medium. Upon quantification, MR image and parameters correlated well to cell seeding densities and alkaline phosphatase activities of various TE constructs. In conclusion, MR can effectively detect the biochemical cascades of osteogenic differentiation of TE constructs and may be a promising, noninvasive monitoring system to provide three-dimensional information for bone tissue engineering.

Notes: Reactive oxygen species (ROS) play a major role in recruiting and activating inflammatory cells, inducing angiogenesis, fibroblast proliferation, and collagen synthesis, all processes essential to wound healing. However, the same mediators have been implicated in pulmonary and hepatic fibrosis, vascular occlusion, and necrotic tissue damage. We have previously shown that hyperbaric oxygen caused a prolonged elevation of VEGF and delayed wound healing in a rat model of tissue ischemia. In this study we examine the effect of the ROS scavenger, n-acetylcysteine (NAC), on ischemic wound healing. NAC is a virtually nontoxic free radical scavenger that has been used in many experimental studies to block ROS-mediated signaling.Methods:  Male Sprague-Dawley rats underwent creation of the ischemic flap previously validated by this laboratory. This model provides two ischemic and two nonischemic excisional wounds. Rats were treated daily with HBO for 90 minutes at 2.4 atm, HBO plus 150 mg/kg NAC intraperitoneal, or control (neither HBO nor NAC). Wounds were analyzed for surface area, lactate, and VEGF.Results:  The HBO/NAC-treated animals had markedly impaired healing of their ischemic wounds at day 7 (0.105 ± .005 cm vs. 0.068 ± .002 cm for ctl and 0.064 ± .006 for HBO) and a twofold elevation of VEGF (120 pg/mg protein vs. 60 pg/mg protein). Lactate levels were not altered by NAC treatment and non-ischemic wounds showed no difference in healing, lactate, or VEGF.Conclusion:  Reactive oxygen species are required for wound healing. Our initial hypothesis was that the delay in wound closure with HBO treatment was due to excess ROS and that NAC would improve healing. The finding of elevated VEGF and delayed wound healing in the presence of a potent free radical scavenger suggests that blocking ROS signaling prevents the normal mitogenic response to VEGF. Additional studies to examine the VEGF receptor and tyrosine kinase activation will further elucidate the mechanism of ROS signaling in response to HBO treatment.

Notes: The Thermage system is a radiofrequency (RF) device designed to treat human skin. The Thermage ThermaCool TC™ is a noninvasive, nonablative, nonlaser system that combines cryogen cooling and radiofrequency (RF) energy to volumetrically heat dermal and subdermal tissue. Thermage treatment causes collagen contraction resulting in immediate skin tightening. It has also been clinically shown to tighten skin over time. This long-term tightening effect is believed to result from the body’s wound healing response. We performed studies in a juvenile pig to characterize the wound healing response after ThermaCool treatment in order to elucidate the cellular and molecular events resulting from treatment. Histological markers of the wound healing process were examined over a 1-month period. Collagen gene expression was examined by quantitative PCR.Macrophages and mast cells are important inflammatory cell types that stimulate fibroblast recruitment, collagen synthesis and blood vessel growth. Three to 5 days following treatment, macrophages and mast cells were present in the dermis. By 28 days, both increased dermal blood vessel density and epidermal thickness was observed. Collagen I and collagen III genes were expressed at elevated levels by 21 days posttreatment.ThermaCool™ treatment stimulates dermal remodeling by initiating a wound-healing response. This study demonstrates that thermage treatment results in the recruitment of important cellular mediators of wound healing and increased collagen synthesis in an animal model. These events are presumed to be responsible for dermal remodeling and skin tightening over time in human subjects. Because the Thermage device noninvasively delivers heat energy, it may be a unique tool for the examination of aseptic thermally induced wound models.

Notes: Induced organ regeneration is de novo synthesis of a physiological, or nearly physiological, organ at the same anatomical site as the organ that is being replaced. Regeneration of skin, peripheral nerves, and the conjunctiva have been accomplished using nothing more than biologically active scaffolds (regeneration templates) seeded with epithelial cells; devices for regeneration of the first two organs are in clinical use. Templates appear to function by blocking contraction well as by acting as temporary configurational guides for synthesis of new stroma that resembles that of the organ under replacement. The combined evidence supports a theory which predicts that selective blocking of the adult healing response uncovers part, at least, of the latent fetal response to injury and, in the presence of the appropriate scaffold, eventually leads to organ regeneration. An independent theory suggests that loss of regenerative potential, which is observed during the mammalian fetal-adult transition, is associated with simultaneous acquisition of individual immunocompetence.

Notes: Fibroblast-collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)-stimulated collagen matrix contraction is independent of Rho kinase whereas platelet-derived growth factor (PDGF)-stimulated contraction is Rho kinase dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA- and PDGF-stimulated fibroblast-collagen matrix contraction. Fibroblasts whose MLC kinase was knocked down using siRNA were used to measure matrix contraction in the presence of LPA or PDGF with or without the Rho kinase inhibitor added. The extent of contraction of MLC kinase-silenced cells was not detectably different from control cells. Other experiments were carried out to test the effects of LPA and PDGF on MLC phosphorylation with and without Rho kinase inhibitor. After 15 min of growth factor stimulation, levels of diphosphorylated MLC were highest in cells in LPA-containing medium and lowest in PDGF containing medium. Prior addition of Rho kinase inhibitor markedly reduced phosphorylation in every case. These observations suggested that stimulation of collagen matrix contraction required neither growth factor stimulation of MLC phosphorylation nor MLC kinase.

Notes: Although the pathogenesis of chronic ulcer still remains undefined, one of the characteristics is poor granulation of wound bed, in other words, disruption of wound angiogenesis. Experimental studies have shown that bone marrow (BM) derived endothelial progenitor cells take part in postnatal neovascularivation in cutaneous wound healing. BM cells also contain mesenchymal stem/progenitor cells with pluripotency differentiating into myofibroblasts and fibroblasts. We treated nonhealing leg and foot ulcers sustained over one year by topical transplantation of autologous fresh unfractionated BM-impregnated collagen sponge (BMiCS). In all patients, the treatments led to rapid generation of well-vascularized granulation tissue. All of the wounds were healed up completely with conservative treatment or skin grafting. We suggest that direct transplantation of autologous bone marrow cells on the wound may represent a novel procedure for regeneration of disrupted wound healing on recalcitrant cutaneous ulcers.

Notes: Aim:  Omental implantation, a surgical procedure in which a perforated gastric or duodenal ulcer is repaired by drawing and implanting a portion of the omentum into the digestive tract, accelerates ulcer healing and inhibits ulcer recurrence compared with omental patch from clinical results. To clarify these mechanism and differences, we investigated ulcer healing in two groups.Methods:  In two groups of rats in which acetic acid-induced gastric ulcers were perforated. Omental implantation was used for repaired in one group and omental patch was employed in the other group. Basic fibroblast growth factor (bFGF) mRNA-positive cells were identified and localized by in situ hybridization. Fibroblast growth factor receptor (FGFR)-positive cells were identified and localized by immunohistochemical analysis.Results:  Antiinflammatory and angiogenic activity and accelerated collagen synthesis were seen in the omental implantation group. BFGF mRNA-positive cells (macrophages, fibroblasts, and endothelial cells) and FGFR-positive cells were seen within the omentum, resulting in abundant collagen production and rapid epithelial regeneration. In the omental patch group, extensive neutrophilic infiltration and ulcer recurrence were seen. Few bFGF mRNA-positive cells and FGFR-positive cells were seen within the omentum, resulting to inhibit omentum becoming to be granulation tissue and ulcer healing.Conclusions:  These results indicated that omental implantation accelerated ulcer healing, and the presence of bFGF mRNA and FGFR played a significant role in this phenomenon.

Notes: Aim:  Most acute phase pressure ulcers have necrotic tissue. Choices to treat such ulcers are controversial. The aim of this study is to help produce a guideline to treat acute phase pressure ulcers with necrosis.Methods:  Records of patients who acute phase ulcers with necrosis were retrospectively reviewed and their primary treatments and outcomes were studied. Ninety-seven ulcers of 86 patients for 3.5 year period were included. Pictures of all ulcers were taken throughout the treatment periods.Results:  Thirteen Stage II ulcers with black or yellow necrosis were treated with hydrocolloid dressings. All of them had excellent results. Forty of 76 Stage III ulcers had yellow necrosis. Hydrocolloid dressings were selected for 24 of them. Two of them became worth. Silver sulfadiazine cream was used for 5 ulcers and the results were relatively good. Fifteen of 36 ulcers with black necrosis were treated with hydrocolloid dressings. Two of them became worth. Thirteen ulcers with black necrosis were resected surgically at the beginnings of the treatments. One of them bled and became worth.Only one of eight Stage IV ulcers had yellow necrosis and it was treated with hydrocolloid dressing and result was good. Remaining five ulcers with black necrosis were resected with good results.Conclusions:  The primary choice for treating Stage II pressure ulcers with any type of necrosis is hydrocolloid dressings. We recommend hydrocolloid dressings for Stage III and IV ulcers without local symptoms of infection. Then after providing autolysis, surgical debridement should be done. If the patient is malnourished or is under poor pressure release, silver sulfadiazine cream may be recommended.

Notes: Aim:  In the present study, expression of members of the fibroblast growth factor (FGF) family and early growth response gene 1 (Egr-1), known as a transcription factor, was analyzed by reverse transcription polymerase chain reaction (RT-PCR).Methods:  After the mouse fetal small intestine was divided into single cells with collagenase-disperse, the cells were cultured. After reorganized small intestine were resected with a knife, expression of mRNAs from FGF family and Egr-1 was analyzed by RT-PCR.Results:  The wound surface was covered with epithelial cells by 24 hours after resection. Among members of the FGF family, mRNAs from FGF-1, -2, -5, and -7 were expressed. Egr-1 was expressed as early as 15 minutes after resection. Egr-1 is thought to be a transcription factor that induces the expression of FGF-2, other than factors involving wound healing, such as PDGF and TNF-a.Conclusions:  In the present study, it was determined that this structure is a potential model for studying the roles of transcription factor and growth factors expressed in association with damage and wound healing.

Notes: Aim:  Trefoil factor family (TFF) peptides are known to facilitate wound healing in gastric mucosa. However, the regulatory mechanisms of gastric TFF expression are not fully understood yet. In this study, we examined the effect of TNF-α on TFF1 and TFF2 expression in gastric epithelial cells.Methods:  MKN45 cells were used. TFF mRNA expression was analyzed by real-time quantitative RT-PCR. Promoter sequences of TFF1 gene (−956 to +36) and TFF2 gene (−912 to +24) were inserted into pGL3 vector and reporter gene assays were performed. NF-κB activity was monitored by using a NF-κB responsive element-driven reporter vector.Results:  (1) TNF-α(0.1–30 ng/ml) down-regulated TFF1 and TFF2 mRNA expression in a dose-dependent manner. (2) Reporter gene assays also confirmed the down-regulation of TFF1 and TFF2 gene transcription by TNF-α. (3) TNF-α activated NF-κB. (4) Overexpression of dominant negative IκBα prevented both TNF-α-induced NF-κB activation and TNF-α-induced down-regulation of TFF expression.Conclusions:  TNF-α down-regulates gastric TFF expression through NF-κB pathway, suggesting that TFF expression is sensitive to inflammatory stimuli.

Notes: Purpose:  Gastrointestinal (GI) damages associated with multiple organ failure is a fatal complication. The injured function of GI tract causes malnutrition and the bacterial translocation. So the functional recovery of GI tract is quite important for treating such cases. In the present study, we investigated whether basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) could recover the injured functions of cultured intestinal cells.Method:  Human small intestinal cells were cultured in a 10% FCS added DMEM. The cellular damages were induced by the treatment with various concentrations of cell permeable C6-ceramide (0–20 M). Growth factors (10 ng/ml bFGF or 50 ng/ml HGF) were added to the medium simultaneously or 2 days after the beginning of the ceramide treatment. After 4 days of the treatment, cell growth and apoptosis induction were investigated.Result and Conclusion:  Ceramide inhibited cell growth and induced apoptosis in a dose-dependent manner. Both growth factors significantly improved the cell growth and reduced apoptosis. These effects were also observed when the growth factor treatment was delayed 2 days after the ceramide treatment. It was found that bFGF was more significant than HGF on these protective effects. It is suggested that growth factor treatment may be a useful therapeutic option to improve GI tract dysfunction.

Notes: Introduction:  Many patients receiving heart surgery have extensive operation scars on the skin surface of the chest wall and lower leg. In some cases there are long lasting open wounds in the lower leg after vein harvesting. Some patients complain of pain and itching and we can recognize hypertrophic or extensive scars, which we have to treat.Case and result:  We treated 16 patients (11 men and 5 women) ranging in age from 47 to 86. They were receiving many drugs such as anticoagulants, thus wound healing was delayed. In these cases, we choose not to resect tissue but apply ointment to remove necrotic tissue, and prepare good granulation tissue for smooth epithelization. Then, we employ prophylactic measures against hypertrophic scar formation such as tape containing steroid, silicon gel sheets and skin tapes as well oral medicine (toranilast) for the same purpose. Because of they have had to continuously take many oral medicines, these patients often do not want to take additional oral medicine. This is one point of difference in the treatment of heart surgery patients and patients with other types of wounds.

Notes: Four off-loading devices used for the treatment of diabetic forefoot ulcers were compared: a custom-molded insole shoe, a cast MABAL shoe, a prefabricated pneumatic walking brace, and a bivalved total contact cast (BTCC). It was anticipated that all devices would significantly reduce forefoot plantar pressure compared with a control shoe. Sixteen healthy volunteers participated. Plantar pressures were evaluated using the Pedar system, while walking speed was determined using an optoelectric timer. Peak pressure (PP) of ten plantar areas and pressure–time integral for the first metatarsal area were analyzed statistically using a repeated measures analysis of variance. Forefoot pressures were significantly reduced compared with the control shoe for all devices (p〈0.001). PP was most reduced (by 65.8%) for the BTCC, and pressure–time integral was most reduced for the BTCC and cast MABAL shoe. Small but significant differences between devices in walking speed (p〈0.05) could not explain the substantial PP differences between the different devices. Although all off-loading devices resulted in a significant reduction of forefoot plantar pressure, devices differed significantly in the magnitude of pressure reduction. Further research will have to reveal the level of off-loading sufficient for effective treatment of diabetic ulcers.

Notes: Hypertrophic scarring and graft contracture are major causes of morbidity after burn injuries. It is well established that application of a split-thickness skin graft reduces scarring and contraction, and cultured epithelial autografts have a similar effect. To investigate the influence of keratinocytes on fibroblast proliferation and fibronectin synthesis, we used an in vitro separated co-culture model in which epithelial sheets were cultured above fibroblast monolayers without physical contact. We also investigated the response of fibroblasts to keratinocyte-conditioned medium (KCM) obtained from confluent and subconfluent keratinocyte monolayers. Both cultured epithelial sheets, composed of adherent fully confluent keratinocytes, and their conditioned medium, reduced fibroblast proliferation. However, KCM from subconfluent keratinocytes stimulated fibroblast proliferation at low concentrations while inhibiting it at higher concentrations, indicating that keratinocytes can produce both mitogenic and growth-inhibiting factors for fibroblasts. KCM, but not epithelial sheet co-culture, also inhibited fibroblast fibronectin synthesis. This indicates regulation of fibroblast phenotype by soluble factors released by the keratinocyte and also suggests that there is a dialogue between keratinocytes and fibroblasts with respect to fibronectin production. We conclude that this separated co-culture model is a simple way to study epithelial/mesenchymal communication particularly with respect to the role of the fibroblast in wound healing.

Notes: A central question in cell biology is how cells become senescent. After a finite number of cell divisions, normal cultured human cells enter a state of irreversible growth arrest, termed “replicative senescence.” Alternatively, oxidative stress in the form of hydrogen peroxide (H2O2) can render human dermal fibroblasts (HDFs) nonproliferative and quiescent, a phenomenon known as stress-induced premature senescence (SIPS). Although critical to the understanding of the pathophysiological basis of many diseases, there is no research to date that has simultaneously examined the interactions between age, oxidative stress, and SIPS. Therefore, the goals of this study were to examine in concert the interactions between these three factors in primary HDFs, and to test our central hypothesis that aging lowers the ability of primary HDFs to respond to oxidative stress. Our data provide, for the first time, evidence that aging dramatically reduces the capacity of primary HDFs to respond to the challenge of hydrogen peroxide. Specifically, aged HDFs showed decreased cell viability, decreased phosphorylation (activation) of pro-survival kinases (Akt and ERK 1/2), and increased entrance into a senescent state when compared with their younger counterparts. Another important conclusion of this study is that blockade of transforming growth factor-β1 had a pronounced “rescue effect” in the aged, preventing entrance of HDFs into cellular senescence.

Notes: Venous leg ulcers are common and cause considerable morbidity in the population. As healing may be slow or may never be achieved, ulcers create persistent and substantial demands on clinical resources. Great efforts have been made to accelerate tissue repair in chronic venous leg ulcers with limited success. This may at least be partly due to the limited knowledge on the microenvironment of chronic wounds. In fact, the tremendous impact of the microenvironmental conditions on the outcome of wound healing has increasingly become apparent. Oxidative stress as a consequence of an imbalance in the prooxidant-antioxidant homeostasis in chronic wounds is thought to drive a deleterious sequence of events finally resulting in the nonhealing state. The majority of reactive oxygen species are most likely released by neutrophils and macrophages and to an unknown extent from resident fibroblasts and endothelial cells. As the inflammatory phase does not resolve in chronic wounds, the load of reactive oxygen species persists over a long period of time with subsequent continuous damage and perpetuation of the inflammation. In this article, we will critically discuss recent findings that support the role of oxidative stress in the pathophysiology of nonhealing chronic venous leg ulcers.

Notes: Transdifferentiation of hepatic stellate cells into proliferative and fibrogenic myofibroblasts is a critical event in the development of liver fibrosis. The causes of HSC transdifferentiation are not well understood, although the extracellular matrix has been implicated as one possible factor. We hypothesized that HSC transdifferentiation is determined by the mechanical properties of the surrounding matrix, and that the diseased liver demonstrates an increased elastic modulus that contributes to the perpetuation of HSC transdifferentiation and the progression of fibrosis. To test this hypothesis in an in vitro system, primary rat HSC were plated on inert polyacrylamide supports of variable but precisely defined elastic modulus ranging from 100 to 12 K Pa. These were coated with different matrices (type I collagen, fibronectin, or Matrigel™) in layers thin enough not to alter the mechanical properties of the system. As determined by morphology, degree of cell spreading, and expression of α-smooth muscle actin, cells became progressively transdifferentiated as substrate stiffness (elastic modulus) increased, independent of the chemical identity of the coating matrix. The degree rather than speed of HSC transdifferentiation correlated with substrate stiffness, with cells cultured on supports of intermediate stiffness adopting stable intermediate phenotypes. These changes were independent of the addition or removal of TGF-β. This demonstrated in an in vitro system that HSC transdifferentiation is primarily a function of the physical rather than the chemical properties of the substrate. As a preliminary to determining the role of stiffness in vivo, we determined the elastic modulus of normal and progressively fibrotic rat liver. Rats were injected twice weekly with carbon tetrachloride or vehicle, and were sacrificed in groups of 7–8 (2 controls and 5–6 CCl4) at time periods ranging from four days to ten weeks after the first injection. Control rats demonstrated a G’(elastic modulus) of 460 +/− 25 Pa. The mean elastic modulus of CCl4-treated livers increased progressively, beginning at the four day point, 525 +/− 39 Pa, and reaching 1617 +/− 170 Pa at 10 weeks. These values are consistent with the values at which we saw intermediate degrees of HSC transdifferentiation in vitro, and suggest that alterations in liver stiffness are a key factor driving the progression of fibrosis.

Notes: The remodeling process in the lung of asthmatic patients results in a deposition of connective tissue and extracellular matrix components (ECM), which has been described as a subepithelial fibrosis. Fibroblasts, with the phenotypic appearance of myofibroblasts, are considered as the key source of the ECM in the fibrotic tissue. Furthermore, has the ECM molecule biglycan shown to correlate to the hyperactivity of the lung of asthmatic patients. In this study, we report the novel finding of a stretched fibroblast phenotype from bronchoalveolar lavage fluid (BALF) in 30% of the asthmatic subjects (n = 13). No fibroblasts were obtained in BALF from any of the nonasthmatic control subjects (n = 17). These BALF fibroblasts with several characteristics of a myofibroblast displayed increased migratory capacity, accompanied by an induced expression of the small GTPases RhoA and Rac1, when compared to myofibroblasts from corresponding lung biopsies. Data also shows that patients with BALF myofibroblast also have more cells in their lung tissue near to the basement membrane that stain for markers for mesechymal progenitor cells. Moreover, the BALF-myofibroblasts showed an increase in production of several types of proteoglycans like biglycan. By adding biglycan to normal fibrobalsts it is further possible to mimic the migratory type of myofibroblast found in BALF. To study differences in protein expression pattern between the two phenotypes, we used a gel-based proteomic technique in combination with MALDI-TOF mass spectrometry. BALF-myofibroblast displayed an increase in heat shock protein 20 (HSP20) and myofibroblasts from biopsies displayed an increase in chloride intracellular channel protein (CLIC) and cofilin. These proteins are all known to regulate cell migration by interacting with the actin cytoskeleton. In summery, data demonstrates that biglycan and mesenchymal progenitor cells might have an important role in the early -remodeling process in asthmatic patients and are potential future biomarkers for sub-epithelial fibrosis formation.

Notes: Common Achilles tendon ruptures are usually not fixed by absorbable sutures due to limitations in their strength properties. Modern technology has made it possible to develop bioabsorbable sutures with prolonged strength retention. To evaluate histologically tissue reactions and biodegradation of poly-L/D-lactide (PLDLA) sutures implanted in Achilles tendon of rabbits. Fifteen rabbits were operated on and killed within a time schedule of 2, 6 and 12 weeks, with five rabbits in per period. PLDLA monofilament sutures (Tampere University of Technology, Tampere, Finland) were implanted inside the rabbit medial gastrocnemius tendon for biocompatibility testing. Polyglyconate (4.0) monofilament sutures (Maxon®, Cyanamid of Great Britain Ltd., Gosport, UK) with the same diameter were implanted in the contralateral tendon. The histology was studied in hard-resin embedded samples and the thickness of the encapsule membrane was determined histomorphometrically.PLDLA have significantly smaller capsule formation around each fiber in tendon than Maxon® sutures. The mean capsule thicknesses around PLDLA sutures was 5.26, 11.66, and 10.63 after 2, 6, and 12 weeks respectively. The mean capsule thicknesses around Maxon® sutures was 13.22, 80.97, and 17.59 after 2, 6, and 12 weeks, respectively. In conclusion, by 12 weeks the PLDLA sutures implanted intratendineously formed thinner fibrous capsule than Maxon® sutures of same diameter. The suture materials were not degraded by 12 weeks.Acknowledgments:  Research funds from the Technology Development Center in Finland (TEKES, Biowaffle Project 40274/03 and MFM Project 424/31/04), the European Commission (EU Spare Parts Project QLK6-CT-2000-00487), the Academy of Finland (Project 73948) and the Ministry of Education (Graduate School of Biomaterials and Tissue Engineering) are greatly appreciated.

Notes: Considering that nitric oxide (NO) plays an important role on cutaneous wound repair, but its effects have not been defined, an inhibitor of NO synthesis (LNAME) was used. Control group received free water and LNAME group received LNAME (20 mg/kg/day) in drinking water. A full-thickness excisional wound was performed (0d), and the animals were sacrificed 7, 14, and 21 days later. Wound contraction was evaluated by lesion surface measurement at 0d, 7d, 14d, and 21d. The blood pressure of all rats was measured in the beginning and at the each time point of the experiment with a plethysmography method. The wound and normal skin surrounding was formol fixed and paraffin embedded. To evaluate the vessels, an immunohistochemistry for α-smooth muscle actin was done and stereological parameters were estimated in superficial and deep dermis. At 7d, 14d, and 21d the blood pressure of LNAME group was higher compared with control group (p 〈 0.001 for all). The wound contraction of LNAME group was slower compared with control group at 14d and 21d (p = 0.004 for both). In superficial dermis, vessels were more dilated in control group compared with LNAME group at 14d and 21d (p = 0.02; p 〈 0.0001, respectively) and longer in control group compared with LNAME group at 7d and 14d (p = 0.043; p = 0.002, respectively). Deep dermal vessels were more prominent and dilated in control group compared with LNAME group at 21d (p = 0.003; p = 0.0007, respectively), but at 7d, vessels were more dilated in LNAME group compared with control group (p = 0.015). In deep dermis, vessels were longer in control group compared with LNAME group at 14d and 21d (p = 0.002; p = 0.046, respectively). Our results show that nitric oxide inhibition affects the wound contraction and the vascularization pattern, mainly in the later phases of skin wound repair.

Notes: Liver myofibroblasts are the key cells of liver fibrogenesis. Recent data show that the serine proteinase thrombin is involved in fibrogenesis through a mitogenic effect on myofibroblasts. The aim of this study was to evaluate the effects of thrombin on the migration of human liver myofibroblasts; another major parameter in fibrogenesis.In a Boyden chamber assay, thrombin dose-dependently (10−10–10−7 M) decreased spontaneous myofibroblast migration down to 49 ± 1% of control values (p = 5.10−10) without affecting cell adhesion or viability. Thrombin effect was blocked by its specific catalytic inhibitor hirudin and could be reproduced by using the proteinase-activated receptor-1 (PAR-1) agonist SFLLRNP. Thrombin also completely inhibited migration when induced by the chemotactic agent platelet-derived growth factor-BB. We then investigated the signaling mechanisms involved. The COX-2 inhibitor NS398 dose-dependently (2.5–10 μM) blunted the inhibitory effect of thrombin on spontaneous migration. However, NS398 did not reverse the inhibitory effect of thrombin on PDGF-BB-induced migration. Phosphatidylinositol 3-kinase (PI3-K) is a major determinant of chemotaxis induced by PDGF. Whereas thrombin did not itself induce PI3-K activity, as shown by the lack of detectable phosphorylation of Akt-1, it inhibited PDGF-BB-induced Akt-1 phosphorylation. This effect was dependent of the Rho/ROCK pathway since it was abolished in the presence of the ROCK inhibitor Y-27632.In summary, thrombin, acting via a proteinase-activated receptor, inhibits human liver myofibroblasts migration. Inhibition of basal migration is dependent on COX-2, while inhibition of PDGF-BB-induced migration involves decreased PI3-K activation via a Rho/ROCK mechanism. We suggest that thrombin could thus stabilize activated myofibroblasts on the site of active fibrogenesis.

Notes: ICR-derived glomerulonephritis (ICGN) mouse is a hereditary model animal for nephrotic syndrome with chronic renal tubulointerstitial fibrosis. In fibrotic disorder, myofibroblastic differentiation plays crucial roles in pathogenesis and is dominantly regulated by the transforming growth factor-beta1 (TGF-β1) signaling system. To reveal the relationships between the pathogenic mechanism of the renal fibrosis and TGF-β1 signaling in ICGN mice, we examined the expression and localization of TGF-β1 signal transducer proteins, TGF-β receptor (TGFR)-I, TGFR-II, Smad2/3 and Smad4, in kidney sections and in primarily cultured renal tubulointerstitial fibroblasts (TIFs). In kidneys of ICGN mice, many TIFs were differentiated to myofibroblastic cells, confirmed as alpha-smooth muscle actin (αSMA)-positive cells, and though no significant increase in production and activation of TGF-β1 were observed in ICGN-TIFs as compared with control ICR-TIFs. The rate of αSMA positive cells in ICGN-TIFs increased during cell culture but not in those of ICR-TIFs. In vitro transcriptional reporter assay for TGF-β1 and Western blotting for TGF-β1 signal transducers revealed that no notable differences in the expression levels of TGFR-I, TGFR-II or Smad2/3 were seen between ICR- and ICGN-TIFs. However, augmented cytoplasmic Smad4 in ICGN-TIFs was observed, and it may cause hypersensitivity against TGF-β1. These results indicate that abnormal cytoplasmic augmentation of Smad4 induces acceleration of TGF-β1 signaling and fibrogenic myofibroblastic differentiation in the renal tubulointerstitial cells of ICGN mice.

Notes: Mechanical stress is a crucial factor for modulating fibroblasts into differentiated myofibroblasts, which are characterized by expression of α-smooth muscle actin (α-SMA). Incorporation of α-SMA into stress fibers generates high contractile activity and drives the formation of “supermature” focal adhesions (FA) that are considerably longer (6–30 μm) compared with “classical” FAs (2–6 μm) of α-SMA-negative fibroblasts. We here show that in turn, supermature FAs control myofibroblast differentiation by communicating the level of extracellular matrix stress to the cytoskeleton. Culture on compliant silicone substrates reduces the size of supermature FAs to that of classical FAs and leads to a concomitant decrease of α-SMA expression. Incorporation of α-SMA into stress fibers requires the formation of FAs longer than 6 μm as demonstrated by plating myofibroblasts on arrays of adhesive islets with dimensions ranging from 1.5 × 2–20 μm and spacing between 2–6 μm, which are created on rigid culture surfaces by means of microcontact printing (μCP). Stretching 6 μm islets on flexible silicone membranes to 8 μm length induces stress fiber formation of α-SMA-EGFP transfected fibroblasts; this was not achieved by applying the same stretch (30%) to cells initially grown on 4 μm islets. By analyzing local deformations created in deformable micropatterned substrates by paxillin-EGFP transfected myofibroblasts, we determined a linear relationship between the size of supermature FAs and local force exertion; hence we determined the minimal tension at individual supermature FAs required for α-SMA recruitment.

Notes: The normal ripening of the human cervix is characterized by rapid degradation of the extracellular matrix, where fibroblasts play an important role. To accomplish this, the fibroblasts undergo phenotypic changes, generally characterized by decreased synthesis of ECM molecules and increased production of degrading enzymes. The hypothesis of this study is that fibroblasts from cervices at different stages of remodeling are of different phenotypes. Fibroblasts were obtained from nonpregnant women, women after 36 weeks of pregnancy and women directly after partus (postpartal) and kept for at least three–four passages to obtain stable conditions prior experiments were performed. The cells were immunostained for differentiation markers, screened for cytokine and MMP expression and production, followed by a global proteomic search. In accordance with in vivo data, a decrease of α-smooth muscle actin and 4-prolyl-hydroxylase was noted by immunohistochemical staining with time of pregnancy. The fibroblasts established from postpartal donors had a three- to fourfold increase of interleukin 6 (IL-6). The production of the chemotactic interleukin 8 (IL-6) was increased even further, five- to sixfold, in postpartal fibroblast cultures. This correlates well to the events in vivo, where IL-6 and Il-8 is produced in high amounts by cervical fibroblast. In addition also the secretion metalloproteinase 1 and 3 are increased threefold in postpartal cultures, which is in agreement with in vivo events. Intermediate amounts of interleukins and metalloproteinases are produced in culture obtained after 36 weeks of pregnancy. A global proteomic approach resulted in regulated proteins involved in Ca-signaling, cytoskeletal mobility and apoptosis. Taken together these data suggest that remodeling of cervix is achieved by a transient activation or recruitment or differentiation of active fibroblasts usually found in inflammatory conditions, characterized by increased MMP and cytokine expression.

Notes: We describe here a new animal model designed to assess the impact of ischemia on wound healing. Among eight patterns of arterial lesion, resection of the external iliac artery down to the femoral artery at the level of the knee was chosen as the reference model, and was performed on the left limb in 24 Wistar rats. The right limb was sham operated and was used as a control. Skin wounds measuring 1.2 × 0.8 cm were created bilaterally on the dorsal aspect of the foot. Blood flow, measured by laser Doppler flowmetry, decreased by over 90% in the ischemic limb after arterial lesion. A significant delay in wound closure was observed with a measurable decrease in wound contraction. Quantification of myofibroblasts by means of α-smooth muscle actin staining showed a significant delay in the appearance and a decrease in the number of myofibroblasts. These findings suggest that decreased wound contraction plays an important role in delayed ischemic wound healing, probably due to reduced myofibroblast development and activity. The model we have developed may also be useful for further investigating other mechanisms of wound healing, for testing pharmaceutical agents and for the study of the impact of pathological conditions such as hypertension or diabetes mellitus.

Notes: Fibroblast to myofibroblast modulation is characterized by expression of α-smooth muscle actin (α-SMA) and represents a crucial step in granulation tissue contraction during wound healing. We have recently demonstrated that myofibroblasts develop cadherin-type cell-cell adherens junctions (AJs), which join stress fibers over the cell membrane. Cadherin expression changes from N-cadherin to OB-cadherin upon TGF-β induced myofibroblast differentiation in culture and coincides with the enlargement of AJs. We here show that myofibroblast AJs exhibit a higher mechanical resistance compared with that of α-SMA-negative fibroblasts by measuring the binding strength between two cells. (1) By separating two suspended cells placed in contact with laser tweezers, we revealed two types of Ca2+-dependent adhesion bonds with different strength. The first bond exhibited a breaking force of 5.5 ± 1.5 nN and existed in both, fibroblasts and myofibroblasts. The strength of the second bond differed between the two cells, breaking at 18.2 ± 0.8 nN in fibroblasts and at 23.0 ± 1.5 nN in myofibroblasts. Measuring the adhesion force between two plated cells by atomic force microscopy after short contact grossly confirmed these observations. Since no cyoskeletal reinforcement was involved in these experimental conditions, we suggest that these differences represent the different cadherins involved in (myo)fibroblast adhesion. (2) Subjecting suspended cells that were attached to plated cells to hydrodynamic forces in a flow chamber demonstrated ∼18% higher adhesion of myofibroblasts over fibroblasts at shear forces of 4 Nm−2. This higher adhesion was reduced to the level of fibroblast attachment by a peptide that inhibits α-SMA-mediated contraction, indicating AJ reinforcement by α-SMA-positive stress fibers. To conclude, high cell-cell adhesion of myofibroblasts appears to be determined by their specific cadherin pattern and by α-SMA-mediated mechanical reinforcement of AJs.

Notes: Fibrocytes are a unique leukocyte subpopulation implicated in wound healing. They are derived from peripheral blood mononuclear cells, display fibroblast-like properties, and synthesize extracellular matrix macromolecules. This study investigated whether fibrocytes are present in healing burn wounds and whether the number of fibrocytes in tissue correlates with the degree of burn injury and the development of hypertrophic scar. Proteins extracted from cultured fibrocytes and nonadherent lymphocytes were found to be similar using two-dimensional gel electrophoresis and quite distinct from those obtained from fibroblasts. However, one protein, identified as leukocyte-specific protein 1 using mass spectrometric peptide mapping, was found in significantly larger amounts in fibrocytes than in lymphocytes but was undetectable in fibroblasts. Double immunostaining with antibodies to leukocyte-specific protein-1 and to the N-terminal propeptide of type I collagen was performed on cryosections of hypertrophic scar, mature scar, and normal skin. Fibrocytes were seen in scar tissue as dual-labeled spindle-shaped cells but were absent from normal skin. Moreover, the number of fibrocytes was higher in hypertrophic than in mature scar tissue. We conclude that fibrocytes, which have been reported to be antigen-presenting cells, are recruited to wounds following extensive burn injury and could potentially upregulate the inflammatory response and synthesize collagen and other matrix macromolecules, thus contributing to the development of hypertrophic scarring.

Notes: Dermal fibroblasts actively contribute to wound healing by migrating to the wound, synthesizing extracellular matrices, and generating mechanical forces within the wound to initiate wound contraction. Fibroblast-seeded collagen gels provide an in vitro model to study wound contraction. The authors are evaluating the role of the adrenergic signaling system in cutaneous wound repair and recently found that β2-adrenergic receptor (β2-AR) activation markedly decreases keratinocyte migration, an essential step in wound reepithelialization. Because the β2-ARs are also expressed on dermal fibroblasts, a study was initiated to determine the effects of β-adrenergic agonists on dermal fibroblast-mediated collagen gel contraction. A β-agonist (isoproterenol) delayed gel contraction in a dose-dependent manner. A β2-AR specific antagonist (ICI 118,551) prevented the delay, indicating that the β2-AR alone mediated the delay. The active cyclic adenosine monophosphate (cAMP) analog also delayed collagen gel contraction, whereas an inactive cAMP analog partially prevented the delay, suggesting that the mechanism for β-AR agonist–mediated delay was partly cAMP-dependent. Identifying and characterizing agents that modulate wound contraction improves understanding of the wound healing process and could result in novel therapeutic strategies for preventing unwanted wound contraction in burn and trauma patients.

Notes: Lucilia sericata larvae, or greenbottle fly maggots, placed within chronic wounds have been observed to remove necrotic tissue and infection. They are also believed to actively promote granulation tissue formation. Interactions between fibroblasts and the surrounding extracellular matrix play a crucial role in tissue formation, influencing fibroblast proliferation, migration, and tissue remodeling. For example, the strength of cell adhesion to surfaces coated with extracellular matrix influences cell motility. L. sericata larval excretory/secretory products having previously been shown to modify fibroblast adhesion to collagen and particularly fibronectin, it was hypothesized that these products would alter fibroblast migration. This was investigated using a two-dimensional in vitro wound assay, time-lapse digital photography, enzyme class-specific substrates and inhibitors, and gel electrophoresis. Results showed that L. sericata excretory/secretory products promoted fibroblast migration upon a fibronectin-coated surface. This was related to the degradation of fibronectin by serine proteinases within maggot excretion/secretions. The presence of a metalloproteinase activity may also have played a role. Thus, a possible mechanism by which maggots enhance tissue formation within wounds may be via the promotion of fibroblast motility, providing for a wider distribution of viable fibroblasts.

Notes: Cell-to–cell interactions between human mesenchymal stem cells and potential adjacent cells such as endothelial cells, dermal fibroblasts, and epidermal keratinocytes was investigated. A modified dual Boyden chamber assay using 8-µm pores revealed a more powerful chemotactic cell migration of human mesenchymal stem cells toward human epidermal keratinocytes than other cells, such as umbilical artery endothelial cells and dermal fibroblasts, during 16 hours of incubation (336.2 ± 52.33, 36.0 ± 11.20, and 62.7 ± 18.16, cells/field, respectively, p 〈 0.01; comparison between endothelial cells and keratinocytes, and fibroblasts and keratinocytes). Scanning electron microscopy showed human mesenchymal stem cell migration through the pores, with endothelial cells, fibroblasts, or keratinocytes in the lower chambers. Mesenchymal stem cell ultrastructural changes occurred, including a larger euchromatin nucleus, when the cells were placed in medium containing 10 percent fetal bovine serum, whereas basic fibroblast growth factor maintained the immature cell morphology for 4 days. Monolayer coculture also showed human mesenchymal stem cell changes in ultrastructural morphology in the vicinity of the epidermal keratinocytes. These data suggest that human mesenchymal stem cells may interact with human epidermal keratinocytes to accelerate wound healing and coverage.

Notes: Introduction:  Diabetic Foot Ulcers (DFU) continue to present a formidable challenge in terms of morbidity and healthcare costs. Increasing evidence ascertains the important role of Matrix Metallo-Proteinases (MMPs) and their tissue inhibitors TIMPS in wound healing. Imbalance of MMPs in the DFU microenvironment has been associated with poor wound healing. Current research is directed toward therapeutic agents that could redress the imbalance of MMPs / TIMPs. Polyhydrated Ionogens (PHIs) formulation is based on metallic ions and citric acid. PHI application aims to positively restore MMP ratios within chronic wounds. This initial multicenter pilot study aimed to investigate the efficacy of the PHI formulation in achieving stable wound closure in recalcitrant DFUs.Methods:  Twenty-nine patients with therapy resistant DFUs of at least 2 cm2 and 3 months duration were treated with PHI formulation in an acetate carrier dressing. Wound debridement, digital imaging, and wound perimeter tracing were performed weekly. Serum samples and punch biopsies were taken from random ulcers for quantitative MMP/TIMP analysis at three time points to assess feasibility of this method. Patient satisfaction was assessed with a questionnaire.Results:  Stable wound closure with high patient satisfaction was achieved in 15 (75%) DFUs. MMP/TIMP ratios within different healing phases were delineated.Discussion:  This pilot study’s encouraging results prompt us to further investigate the PHI efficacy in DFU treatment in a Web-based, multicenter, randomized controlled trial.

Notes: Purpose:  Gene therapy provides a useful method to enhance wound healing, but it has not been attempted in healing of wounds in flexor tendons in the hands, which inherently lack sufficient intrinsic healing capacity. To explore the potential of application of gene therapy to tendon wound healing, we genetically modified tenocytes with the platelet-derived growth factor-B (PDGF-B) and the vascular endothelial growth factor (VEGF) gene and investigated the expression of the genes for collagen production in an in vitro model of the proliferating tenocytes.Method:  Tenocytes were obtained from cultures of rat intrasynovial tendons and randomly distributed to 34 dishes. The tenocytes in dishes in two experimental groups (n = 9, each group) were treated for 12 hours with the plasmid containing the PDGF-B cDNA or the VEGF cDNA and were then cultured for 5 days; tenocytes in the other eight dished received sham vector and the tenocytes in the control dishes (n = 8) did not receive the exogenous gene. Efficiency of the gene transfer was evaluated by detection of the presence of the transgene in the tenocytes by reverse transcription polymerase chain reactions (RT-PCR). Levels of expression of type I and III collagen, and TGF-β genes were determined by quantitative analysis of the products of RT-PCR.Results:  Expression of the type I collagen gene was significantly increased by transfer of the exogenous PFDGF gene to the tenocytes (p 〈 0.001). Transfer of the PDGF-B gene did not affect the expression of TGF-β and the type III collagen genes significantly. Expression of TGF-β gene increased significantly in the cells treated with exogenous VEGF cDNA (p = 0.039). Expression of type I and III collagen genes by tenocytes was minimally affected by transfer of the VEGF gene to the tenocytes and was significantly weaker than that stimulated by PDGF-B gene therapy (p = 0.001). Efficient gene transfer was confirmed by the presence of the PDGF-B gene or the VEGF gene in the tenocytes receiving the transferred genes.Conclusions:  Transfer of exogenous PDGF-B gene significantly enhances expression of the type I collagen gene of the tenocytes. Transfer of exogenous VEGF gene has very limited effects on promotion of collagen production in the proliferating tenocytes. In contrast, transfer of the VEGF gene significantly increases expression of the TGF-β gene. This study suggests that transfer of the PDGF-B gene may offer a novel way of effectively promoting healing of intrasynovial flexor tendons. VEGF gene therapy is not as beneficial as PDGF-B gene therapy to tendon healing and may increase activities of TGF-β that are associated with adhesion formations.

Notes: Tissue ischemia is a common occurrence in many disease processes including chronic wounds, stroke, solid tumors, and myocardial infarction. The application of gene delivery for healing of wounds has demonstrated increasing therapeutic promises in animal models. Adenoviral vectors have been successfully used for gene delivery to the ischemic wound. However, these vectors typically demonstrate short, transient transgene expression while eliciting significant cytotoxic immune response. Adeno-associated viral vectors (AAV) do not have those limitations; however, scant information is available about their transfection efficiency under low-oxygen tension. The goal of this study was to compare AAV vector with adenoviral vector in terms of relative efficiency of gene delivery and cytotoxic immune response in ischemic wounds. Reporter constructs Ad5-LacZ and AAV-LacZ (108 pfu/wound) were injected onto the dermis of rabbit ear prior to creation of ischemic wounds. Wounds were harvested at postoperative day 10. Frozen sections of the wounds were fixed in cold acetone and stained with an in situ β-gal staining kit. Intense expression of β-gal was observed with both vectors; however, transduction rates with AAV vector was approximately 10-fold lower than Adenovirus. Unlike Adenovirus, no noticeable inflammatory cell infiltration was observed with AAV injection. Even when the dosage of AAV was increased to 109 pfu/wound inflammatory cell infiltration remained negligible. Thus our data indicates that both AAV and adenoviral vectors are suitable to use in gene-therapy experiments in ischemic tissues. The particular advantage of AAV is the ability to transfect with higher doses while at lower dose maximal transfection rate seems to be more with Adenovirus.

Notes: Fibroblasts in chronic wounds, and in skin in which ulcers develop, have been reported to show characteristics of senescence, presumably induced by conditions predisposing to chronic wounds or the wound environment. In monolayer culture, secretion of IL-8 declines to zero as the cells approach senescence (at about generations 50–55 in the cells tested). In three-dimensional scaffold-based tissue culture, fibroblasts, at about 30–35 population doublings, upregulate the IL-8 gene by comparison with monolayer cultures and secrete large amounts of IL-8 (30–50 ng/106 cells/day). However, it is inferred that a decline in secretion, similar to that seen in monolayer also occurs in three-dimensional culture and in the senescent fibroblasts of chronic wounds. This will result in decreased influx and activation of neutrophils. The hypothesis is proposed that chronic wounds arise because fibroblasts, following injury to ulcer-prone skin, do not recruit and activate neutrophils sufficiently to prevent bacterial colonization. As the result of the persistent presence of live bacteria, inflammatory cells or keratinocytes in a wound site may be expected to trigger mechanisms to inhibit keratinocyte migration and reepithelialization, leading to chronic ulceration.

Notes: Delayed healing can result for a number of reasons; however, one important factor in the wound healing process is the role of bacteria. An imbalance in the wound bioburden may lead to infection, and thereby severely delay healing. Thus to ensure healing progresses in a timely manner, it is necessary to maintain a healthy microbial balance and prevent any impending infection.Dermal cells such as fibroblasts and keratinocytes generate numerous chemokines and growth factors that regulate the immune response and the phases of healing. While topical antiseptics and antimicrobials can kill bacteria and are therefore used to help manage infection in wounds, they may also be cytotoxic to these important cell types. Cytotoxicity is acceptable when overt clinical infection is present, due to a greater need for bacterial balance. However, clinical signs of infection in chronic wounds may not be present, making it difficult to correctly determine diagnosis and appropriate use of these antimicrobial products. In these cases, a product which addresses both the antibacterial action of topical antiseptics/antimicrobials and their impact on key healing factors, (i.e., cytotoxicity to host cells), could help reduce the incidence of wound infection without impacting the rate of healing.In this study we investigated the effect of silver-ORC and an ORC/collagen matrix containing silver-ORC on human dermal fibroblast proliferation. The antimicrobial properties of the dressing was also investigated, in in vitro studies using common bacterial wound pathogens.While other silver-releasing dressings address the bioburden of nonhealing wounds, their in vitro cytotoxicity suggests that the rate of healing may not be optimal if the presence of infection is questionable. In contrast this in vitro work suggests the ORC/collagen matrix containing silver-ORC is not cytotoxic, but is still capable of controlling wound pathogens, suggesting it may be used on a broad spectrum of wounds independent of their bacterial bioburden.

Notes: Removal of necrotic tissue is essential for wound-bed preparation for the treatment of chronic wounds. Enzymatic debridement appears to be most useful in the removal of eschar when surgical techniques cannot be utilized. The purpose of this study is to determine the efficacy of papain–urea chlorophyllin ointment compared to moist wound care using an infected, acute, porcine full thickness wound model. Twenty full thickness wounds were created on the dorsum of domestic pigs. Following hemostasis, the wounds were contaminated with wound-isolated bacteria then treated with test articles and dressed with moisture-retentive dressing. All the wounds were evaluated 1, 4, 8, 11, 14,18, and 21 days postsurgery for epithelialization and biopsies were obtained for microscopic evaluation. The results showed no adverse effect in reepithelialization in acute wounds when using papain–urea chlorophyllin "ointment compared to moist wound care, TEWL measurements also showed no adverse affects from the treatment. Microscopic examination reveals an increase in the number of keratinocytes present in the epidermis of the treated pigs as well as an increase in the depth of rete pegs. New blood vessel formation was increased at all examined time points in the papain–urea chlorophyllin ointment treated pigs, which was verified with CD31 and von Willebrand Factor staining. VEGF was increased after 1 week in the wound fluid from treated wounds compared to moist control. While the reepithelialization rate does not appear to be inhibited by papain–urea-chlorophyllin combination in acute wounds, the healing appears to be more complete based on the number of keratinocytes present in the epidermis and the extensive rete peg formation. The wounds also appear to have enhanced vascularization which is vital in supplying nutrients to the newly healed wound.

Notes: We previously showed that Calreticulin (CRT), a chaperone of the endoplasmic reticulum, has profound effects on the process of wound healing by increasing both epithelial migration and granulation tissue formation in models of impaired porcine and murine repair. In the present study, using the scratch plate assay as an in vitro model of wound repair, we show that CRT (10−6–10−9 M) induces cell migration/wound closure in human keratinocytes and fibroblasts that is equivalent to EGF (positive control). Furthermore, by an MTS proliferation assay, CRT stimulated cellular proliferation of human keratinocytes (2-fold), fibroblasts (100-fold), and vascular endothelial cells (1.2-fold). Markedly increased cellular proliferation was confirmed by anti-ki67 immunostaining (IHC) which was confined to basal keratinocytes in the CRT-(5 mg/ml) treated porcine and murine wounds during reepithelialization and in numerous cells of the granulation tissue. Also, CRT increased dermal depth and granulation tissue formation between days 6–14 following injury (p 〈 0.05). The effect of CRT on the dermal layer was qualitatively different than PDGF or PBS as there was a remarkably higher number of dermal cells that reconstituted the CRT-treated pig wounds, a more organized pattern of collagen organization by picrosirrius red staining, and greater fibronectin and TGF-β3 by IHC. CRT also increased tensile strength in a rat incisional wound model (p 〈 0.005 @ 21 days). By Mac-138 IHC of porcine wounds, we observed an increase in macrophages in the wound bed compared to PDGF (p 〈 0.013). Since the uptake of apoptotic cells by phagocytes is a CRT-dependent process, this finding suggests a potential role for CRT as a bacteriocidal and/or debriding agent. Unlike PDGF, CRT positively affects both the epithelial and dermal aspects of repair. Our studies suggest that CRT accelerates and improves the rate and quality of wound repair by affecting many cells involved in the repair process.

Notes: The paradigms of surgical wound closure are direct repair, grafts, and flaps. The core subject of plastic surgery, these distinct modalities are a sophisticated art that can reliably close any healthy wound. The caveat is that ordinary surgery implicitly depends on competent wound healing. With pathological wounds, those due to vascular, hematological, immunopathic, and other ulcerogenic diseases, these modalities may not work. The operation is threatened if wound healing is retarded, and incisions and donor sites are subject to morbidity from active disease. Disease and risks may limit potential donor tissues, sustain inflammation and lysis, restrict circulation, or make the patient too ill for elaborate procedures.Integra® collagen-chondroitin matrix has ideal properties for managing chronic pathological wounds: a high-grade artificial skin; survives disease and conditions where grafts die; performs the coverage duties of flaps without donor sites; minimizes nursing care; suppresses inflammation, wound healing, and scar; induces embryonic dermatogenesis. Risk free to the recipient, it is safe where disease and altered anatomy make conventional closure impossible or unsafe. Its indications, use, results, and conceptual basis make it a distinct fourth paradigm of surgical wound closure: in situ tissue engineering. Understanding when a flap should but cannot be used is to understand when Integra should be used.Study:  Using a consistent set of indications and management scheme, Integra was used for chronic pathological wounds in 111 patients (158 individual ulcers, 166 instances of exposed anatomical structures, diverse diagnoses). Success rate was 92% of patients healed; 90% of open structures healed. Inpatient services were nearly eliminated.Integra is not an alternative to conventional repair, grafts, and flaps. As a method of tissue engineering, it is a new and equal paradigm of care with its own indications and contraindications, and a superior safety and success profile. Repair, grafts, and flaps, which require wound healing competency, are best suited for healthy and acute wounds. For many chronic and pathological wounds, in situ matrix-guided histogenesis is the best method, and surgeons must begin working this into their practices.

Notes: The potential to lower destructively high levels of elastase found in chronic wounds with oleic acid-treated cotton wound dressings was assessed and compared with two types of oleic acid-treated occlusive wound dressings. The ability of albumin to bind oleic acid and transfer elastase inhibitory activity from the dressing material to elastase in solution was examined. Cotton, hydrogel, and hydrocolloid wound dressings were treated with oleic acid and tested for their ability to lower elastase activity. Oleic acid–treated cotton gauzes, hydrogels, and hydrocolloid dressings were found to release oleic acid in the presence of albumin in sufficient quantities to inhibit elastase activity. The order of elastase lowering activity with the three oleic acid-formulated wound dressings was cotton gauze 〉 hydrogel 〉 hydrocolloid. Elastase inhibition by oleic acid displaced from gauze followed a dose response profile. In contrast Cathepsin G, when displaced by albumin, was inhibited within a narrow range of oleic acid formulations. The effect of albumin levels representative of the chronic wound on displacement of oleic acid was determined. Solubilization of cotton bound oleic acid by albumin was found to be most effective at 4% albumin. However, there was sufficient solubilization of oleic acid at the 2% albumin levels found in chronic wound fluid to achieve a significant elastase-lowering effect. Albumin promoted solubilization of oleic acid from cotton with a solubility threshold of 27 mg/g cotton gauze. Equivalent lowering of elastase activity was found with 1%, 2%, and 4% albumin concentrations with oleic acid on cotton at 128 mg oleic acid/gram of cotton.

Notes: Introduction:  Currently freeze-dried, gamma-sterilized, or glycerol-preserved amniotic membranes are widely used. However, it is not clear whether this devitalized state is the optimal application form. Therefore within this study the ideal condition for midterm storage of human amniotic membranes was assessed to ensure the availability of vital amniotic membranes, in particular for burn wounds.Methods and Materials:  For this purpose mothers were serologically tested and term placentae were collected and washed. After the amniotic membrane was peeled off and further washed, biopsies were taken for microbiological testing and various storage experiments (different media and temperatures).〈list style="custom"〉• cell culture medium, 37 °C• glycerol, 4 °C• 10% DMSO, −80 °CViability of fresh and stored amniotic membranes was determined with the MTT-based EZ4U- Assay (Biomedica, Vienna, Austria).Results and Discussion:  Best results were obtained while storing the membranes in cell culture medium at 37 °C, whereas storage in glycerol at 4 °C resulted in cell death within the first 4 days.To our knowledge this is the first study investigating the viability of amniotic membrane under different storage conditions. The influence on wound healing is currently under investigation.

Notes: Sepsis remains the single most important cause for multiple organ failure and death following burn injury for which no effective treatment is currently available. Burn injury is associated with immunosuppression which promotes sepsis. Studies from our laboratory have demonstrated that levels of the antimicrobial peptide (AMP), human β-defensin-2, was significantly decreased in burned skin epithelia, absent in burn blister fluid and poorly expressed in lung during inhalation injury.We hypothesize that 1) deficient antimicrobial peptides in burn wound promote progressive colonization of pathogens in burn wounds and 2) replacing active peptide components will restore the antimicrobial activity in burn wounds. We have synthesized several cationic steroid antimicrobials (CSAs) which mimic naturally occurring AMPs functionally. In vitro experiments using one of these synthetic drugs with locally isolated common burn pathogen P. aeruginosa and S aureus demonstrate MIC values of 2.0 ± 0.3 μg/ml and 0.4 ± 0.1 μg/ml, respectively, indicating effectiveness of these compounds against burn pathogens. These drugs were tested for cytotoxicity on cultured human keratinocytes using MTT cell viability assay. Results indicate that these drugs are nontoxic to cultured keratinocytes at the level required for bacterial killing. Since theses drugs are membrane active compounds it will be hard for bacteria to develop resistance and there is a great potential to be developed for clinical treatment.

Notes: Background:  Acinetobacter baumannii has recently emerged as an important hospital-acquired pathogen, especially in surgery, burn, and intensive care units. Due to its ability to develop resistance to antimicrobials, wound infection with A. baumannii is difficult to treat, and can lead to septicemia and even death. Use of appropriate topical antimicrobial agents in these circumstances could be one of the first steps in prevention of A. baumannii wound infection.Objectives:  In this study, we will discuss the in vitro effects of seven common topical antimicrobial creams and dressings on A. baumannii.Methods:  A. Baumannii ATCC# 6919 was subjected to sensitivity tests against mupirocin, silver sulfadiazine, mafenide acetate, a double antibiotic combination of polymyxin and bacitracin, a triple antibiotic combination of Neomycin, bacitracin and polymyxin, and two silver-containing dressings. Zones of inhibition were measured after 24 hours incubation period.Results and Conclusion:  Of the evaluated antimicrobial agents, mafenide acetate was the most efficacious followed by mupirocin, triple and double antibiotic combinations in decreasing order. The silver-containing dressings yielded a lesser zone of inhibition as compared to the previously mentioned, and no zone of inhibition was observed using silver sulfadiazine. Further in vivo studies on the effect of antimicrobial agents against A. Baumannii are necessary to substantiate these findings and determine the potential clinical relevance of these therapies.

Notes: Often chronic wounds have an increased bacterial burden that can impair healing without the classical clinical signs of infection. Silver dressings may provide an alternative topical method to control bacterial burden.The primary aim of this study was to evaluate the effect of 2–4 weeks therapy with the Silver Containing Hydrofiber® dressing on quantitative bacterial burden and clinical improvement in chronic wounds not healing at the expected rate.This was a single centre, four-armed study which included a total of 30 patients with diabetic foot ulcers, leg ulcers, pressure ulcers and miscellaneous wounds that did not fit into any of the above categories. Patients had a baseline quantitative bacterial biopsy and this was repeated at weeks 2 to 4. The wound size was recorded along with a semi quantitative estimate of exudate and the periwound temperatures. Repeat measurements were performed at the follow-up visits and the decrease in wound size calculated. The underlying cause of the ulceration was be treated and corrected. This was followed by application of silver containing hydrofiber® dressing. There was a significant delay in healing of the leg ulcers associated with increased bacterial burden in the quantitative biopsy bacterial burden results at week 0 and healing at week 2. (p = 0.01). Other subgroups had a similar association that did not reach statistical significance. The presence of an increased exudate in the leg ulcers at week two was associated with delayed healing at week 4 (p = 0.05). There was also a significant increase in skin surface temperature of the surrounding skin with an increased quantitative bacterial biopsy of the deep wound compartment for venous, diabetic neurotrophic foot ulcers and pressure ulcers with p values of 0.05, 0.01 and 0.01 respectively. There was no significant decrease in exudate or increased healing of the wounds with the application of the silver hydrofiber dressing in this difficult to heal population. The population studied in this case series had increased bacterial burden in the deep compartment as measured with increased exudate and or an increased temperature of the periwound skin. These patients have an increased bacterial burden in the deep wound compartment that does not respond to topical ionized silver in the dressing studied.

Notes: Despite recent advances in wound care, chronic infected ulcers still remain a huge challenge to the modern society. Nitric oxide has been identified as a mediator for important biological processes such as vasodilation, antimicrobial, and wound repair. The aim of the present study was to explore the antimicrobial properties of exogenous nitric oxide gas (gNO) as well as its effect on skin cells in vitro. To test this, a specialized gNO chamber was designed to expose S. aureus and P. aeruginosa in saline continuously to 200 ppm gNO or air. A colony count was obtained following gNO treatment. Furthermore, fibroblast-populated collagen gel, keratinocytes, and endothelial cells were cultured in DMEM, KSFM, and M199, respectively. Endothelial migration was tested by a 3D Matrigel tubule formation assay. Fibroblast matrix production and keratinocyte differentiation were monitored by mRNA expression of procollagen type I, interstitial collagenase, and involucrin. All cells were exposed to 200 ppm gNO for 8 hours a day for 3 consecutive days. A MTT assay was used to observe cell proliferation. The results revealed 100% bacterial kill following 4 hour exposure to 200 ppm gNO. Interestingly this dose did not exhibit any significant toxic effect on fibroblast, keratinocyte, and endothelial proliferation. Dermal fibroblasts migration out of the collagen gel was not affected by gNO treatment when compared to the control. The keratinocytes in both groups were able to express involucrin, an indicator of cell’s ability to differentiate. The migration and tube formation by endothelial cells within the matrigel was not compromised following gNO treatment. In conclusion, the present study provided evidence for potential application of gNO for reducing bacterial burden in chronic infected wounds without compromising re-epithilialization, proliferation, and angiogenesis in the wound healing process.Acknowledgment:  This study was funded by PulmoNOx Medical Inc.

Notes: Skin substitutes are slowly finding a position in the treatment of burns, scar reconstructions and chronic wounds. Some of the substitutes consist of extracellular matrix replacement material only, such as Integra and Alloderm; some include allogeneic cells (Dermagraft, Appligraf). The ideal skin substitute has not been developed yet, since none of the presently available products can ultimately prevent scar formation.Study of the role of autologous fibroblasts in the healing process might give further insight into the scarring process and eventually lead to improved skin substitutes.We compared wound reepithelialization of experimental wounds treated with proliferating keratinocytes and a dermal substitute with either dermal fibroblasts, adipose tissue derived fibroblasts or no fibroblasts. We also investigated the rate of keratinocyte migration of human skin equivalents cultured in vitro in the presence of dermal or adipose tissue derived fibroblasts.We reached successful wound closure in 8 days with transfer of proliferating keratinocytes on a dermal substitute seeded with dermal fibroblasts. However, the wounds treated with substitutes which contained adipose tissue derived fibroblasts or no fibroblasts at all were not closed even after 21 days.Keratinocytes seeded onto collagen lattices populated with either dermal or fat-derived fibroblasts showed similar findings: a retarded migration and/or proliferation of keratinocytes on the collagen lattices with fat-derived fibroblasts. The collagen lattices populated with fat-derived fibroblasts also showed a marked contraction, up till 50% of the original area.In both models, more alpha-smooth muscle actin positive cells were found in the fibroblast population from adipose origin.We conclude that epidermal regeneration is negatively influenced by the presence of fat-derived fibroblasts in a dermal matrix; possibly, myofibroblasts play a role in this.

Notes: It has been known for several decades that early gestational human fetuses will regenerate cutaneous wounds resulting in a scarless repair. In postnatal humans, myofibroblasts are known to play a central role in wound healing and in the pathology of fibrosis. The role of the profibrotic growth factor TGF-β1 in human fetal fibroblasts remains unclear, with recent data now showing that TGF-β1 is present in fetal wounds but for shorter periods. We have therefore examined the effects of TGF-β1 on fibroblasts isolated from early human fetuses (〈14week EGA). We have identified that stimulation with 5 ng/ml TGF-β1 induces a significant proportion of human fetal fibroblasts to differentiate into myofibroblasts (approximately 40%), as identified by staining of α-smooth muscle actin (n = 5). Interestingly, this response was earlier (peaking at day 2–3) and more transitory (over by day 4–5) than that seen with postnatal dermal fibroblasts (peaking at day 6–8 and dropping at day 10). We examined the effects of blocking various intracellular pathways involved in TGF-β1 signalling. The early and transitory myofibroblast induction seen in fetal cells is blocked by an inhibitor of p-JNK (n = 4; p =〈 0.05). In contrast, the later and more prolonged induction of myofibroblasts seen in postnatal cells was not affected by inhibition of p-JNK. Despite the fact that fetal fibroblasts do indeed differentiate into myofibroblasts they were further shown to exhibit differential behavior to postnatal myofibroblasts with respect to TGF-β1 stimulation of collagen expression, producing significantly less soluble collagen (approximately 50%, n = 4) after 24 or 48 hours of treatment with TGF-β1 in serum-free media. These results appear to indicate a role for TGF-β1 during cutaneous wound healing in the early fetus and show that the response of fetal cells to this important profibrotic cytokine is rapid and more controlled, being short lived. It is possible that a prolonged response to this cytokine may lead to the overblown nature of postnatal wound healing, resulting in fibrosis.

Notes: Cellular contraction, defined as the force cells exhibit on the extracellular matrix, plays a significant role in dermal tissue. To study the contractility of cells in vitro, we have developed a new patented device named GlaSbox®“Growing Lattice Study Box”. It consists of a cell chamber composed with 8 rectangular wells (26 × 33 mM) in which lattices develop. Two opposite silicon beams hang down into each well. The lattice is attached to this sensor through a grid directly etched on the lower part of the beams. A strain gauge is deposited at the beam surface, and connected to form a Wheastone bridge. The strain gauges signal output is amplified then converted and collected by a computer which includes an acquisition card and a specific program giving directly the forces in real time. The significant advantages of the GlaSbox® are:〈list style="custom"〉– the absence of any interface between the collagen lattice and the beams which maintain it stretched,– its ability to test eight lattices simultaneously,– the low volumes of cells and collagen required.To demonstrate the applicability of this system, contractile forces of human fibroblasts from different skin conditions were measured such as chronic venous leg ulcers and striae distensae. These fibroblasts were found to produce an average force of 1.10−8 N/cell and have greater contractile capacity than normal fibroblasts (Viennet et al., J Wound Care 2004, in press). The GlaSbox® is a useful tool to study the contractility of dermis equivalent composed of various fibroblast lines and, the effect of different active principles or even growth factors (e.g., TGF-β) on cellular contraction. In addition, it can be used to examine the relationship between cellular contraction, cell proliferation, and collagen production, which is important for the understanding of the mechanism of scar tissue formation.

Notes: A petrogenetic grid in the model system CaO–FeO–MgO–Al2O3–SiO2–H2O is presented, illustrating the phase relationships among the minerals grunerite, hornblende, garnet, clinopyroxene, chlorite, olivine, anorthite, zoisite and aluminosilicates, with quartz and H2O in excess. The grid was calculated with the computer software thermocalc, using an upgraded version of the internally consistent thermodynamic dataset HP98 and non-ideal mixing activity models for all solid solutions. From this grid, quantitative phase diagrams (P–T pseudosections) are derived and employed to infer a P–T path for grunerite–garnet-bearing amphibolites from the Endora Klippe, part of the Venetia Klippen Complex within the Central Zone of the Limpopo Belt. Agreement between calculated and observed mineral assemblages and garnet zonation indicates that this part of the Central Zone underwent a prograde temperature and pressure increase from c. 540 °C/4.5 kbar to 650 °C/6.5 kbar, followed by a post-peak metamorphic pressure decrease. The inferred P–T path supports a geotectonic model suggesting that the area surrounding the Venetia kimberlite pipes represents the amphibolite-facies roof zone of migmatitic gneisses and granulites that occur widely within the Central Zone. In addition, the P–T path conforms to an interpretation that the Proterozoic evolution of the Central Zone was controlled by horizontal tectonics, causing stacking and differential heating at c. 2.0 Ga.

Notes: A review and reinterpretation of previous experimental data on the deformation of partially melted crustal rocks reveals that the relationship of aggregate strength to melt fraction is non-linear, even if plotted on a linear ordinate and abscissa. At melt fractions, Φ 〈 0.07, the dependence of aggregate strength on Φ is significantly greater than at Φ 〉 0.07. This melt fraction (Φ = 0.07) marks the transition from a significant increase in the proportion of melt-bearing grain boundaries up to this point to a minor increase thereafter. Therefore, we suggest that it is the increase of melt-interconnectivity that causes the dramatic strength drop between the solidus and a melt fraction of 0.07. We term this drop the ‘melt connectivity transition’ (MCT). A second, less-pronounced strength drop occurs at higher melt fractions and corresponds to the breakdown of the solid (crystal) framework. This is the ‘solid-to-liquid transition’ (SLT), corresponding to the well known ‘rheologically critical melt percentage’. Although the strength drop at the SLT is about four orders of magnitude, the absolute value of this drop is small compared with the absolute strength of the unmelted aggregate, rendering the SLT invisible in a linear aggregate strength v. melt-fraction diagram. On the other hand, the more important MCT has been overlooked in previous work because experimental data usually are plotted in logarithmic strength v. melt-fraction diagrams, obscuring large strength drops at high absolute strength values. We propose that crustal-scale localization of deformation effectively coincides with the onset of melting, pre-empting attainment of the SLT in most geological settings. The SLT may be restricted to controlling flow localization within magmatic bodies, especially where melt accumulates.

Notes: Contact metamorphism caused by the Glenmore plug in Ardnamurchan, a magma conduit active for 1 month, resulted in partial melting, with melt now preserved as glass. The pristine nature of much of the aureole provides a natural laboratory in which to investigate the distribution of melt. A simple thermal model, based on the first appearance of melt on quartz–feldspar grain boundaries, the first appearance of quartz paramorphs after tridymite and a plausible magma intrusion temperature, provides a time-scale for melting. The onset of melting on quartz–feldspar grain boundaries was initially rapid, with an almost constant further increase in melt rim thickness at an average rate of 0.5–1.0 × 10−9 cm s−1. This rate was most probably controlled by the distribution of limited amounts of H2O on the grain boundaries and in the melt rims.The melt in the inner parts of the aureole formed an interconnected grain-boundary scale network, and there is evidence for only limited melt movement and segregation. Layer-parallel segregations and cross-cutting veins occur within 0.6 m of the contact, where the melt volume exceeded 40%. The coincidence of the first appearance of these signs of the segregation of melt in parts of the aureole that attained the temperature at which melting in the Qtz–Ab–Or system could occur, suggests that internally generated overpressure consequent to fluid-absent melting was instrumental in the onset of melt movement.

Notes: New eclogite localities and new 40Ar/39Ar ages within the Western Gneiss Region of Norway define three discrete ultrahigh-pressure (UHP) domains that are separated by distinctly lower pressure, eclogite facies rocks. The sizes of the UHP domains range from c. 2500 to 100 km2; if the UHP culminations are part of a continuous sheet at depth, the Western Gneiss Region UHP terrane has minimum dimensions of c. 165 × 50 × 5 km. 40Ar/39Ar mica and K-feldspar ages show that this outcrop pattern is the result of gentle regional-scale folding younger than 380 Ma, and possibly 335 Ma. The UHP and intervening high-pressure (HP) domains are composed of eclogite-bearing orthogneiss basement overlain by eclogite-bearing allochthons. The allochthons are dominated by garnet amphibolite and pelitic schist with minor quartzite, carbonate, calc-silicate, peridotite, and eclogite. Sm/Nd core and rim ages of 992 and 894 Ma from a 15-cm garnet indicate local preservation of Precambrian metamorphism within the allochthons. Metapelites within the allochthons indicate near-isothermal decompression following (U)HP metamorphism: they record upper amphibolite facies recrystallization at 12–17 kbar and c. 750 °C during exhumation from mantle depths, followed by a low-pressure sillimanite + cordierite overprint at c. 5 kbar and c. 750 °C. New 40Ar/39Ar hornblende ages of 402 Ma document that this decompression from eclogite-facies conditions at 410–405 Ma to mid-crustal depths occurred in a few million years. The short timescale and consistently high temperatures imply adiabatic exhumation of a UHP body with minimum dimensions of 20–30 km. 40Ar/39Ar muscovite ages of 397–380 Ma show that this extreme heat advection was followed by rapid cooling (c. 30 °C Myr−1), perhaps because of continued tectonic unroofing.

Notes: High-grade gneisses (amphibolite–granulite facies) of the Namche Barwa and Gyala Peri massifs, in the eastern Himalayan syntaxis, have been unroofed from metamorphic depths in the late Tertiary–Recent. Rapid exhumation (2–5 mm year−1) has resulted in a pronounced shallow conductive thermal anomaly beneath the massifs and the intervening Tsangpo gorge. The position of the 300 °C isotherm has been estimated from fluid inclusions using CO2–H2O immiscibility phase equilibria to be between 2.5 and 6.2 km depth below surface. Hence, the near-surface average thermal gradient exceeds 50 °C km−1 beneath valleys, although the thermal gradient is relatively lower beneath the high mountains. The original metamorphic fluid in the gneisses was 〉90% CO2. This fluid was displaced by incursion of brines from overlying marine sedimentary rocks that have since been largely removed by erosion. Brines can exceed 60 wt% dissolved salts, and include Ca, Na, K and Fe chlorides. These brines were remobilized during the earliest stages of uplift at 〉500 °C. During exhumation, incursion of abundant topography-driven surface waters resulted in widespread fracture-controlled hydrothermal activity and brine dilution down to the brittle–ductile transition. Boiling water was particularly common at shallow levels (〈2.5 km) beneath the Yarlung Tsangpo valley, and numerous hot springs occur at the surface in this valley. Dry steam is not a major feature of the hydrothermal system in the eastern syntaxis (in contrast to the western syntaxis at Nanga Parbat), but some dry steam fluids may have developed locally.

Notes: Abstract  Objective: To identify the relationship of work stress and family stress to the health of women in Korea. Design: Cross-sectional study. Sample: Three hundred and thirty-one married women working in 14 manufacturing companies in Korea. Methods: Subjects responded to a questionnaire that included items on work stress, family stress, social support, and general characteristics. Perceived health status (PHS) was assessed with the Short Form-36. Results: There was a significant positive relationship between social support and PHS, but significant negative relationships were found between PHS and work stress as well as family stress. Hierarchical multiple regression analysis explained the health status of married working women by four categories: personal, work related, family related, and social support, and accounted for 45.4% of the variance. When family-related factors were added to the model, the power of explanation was increased by 17.9% compared with the explained variance. Family stress was a major variable not only for explaining the variance but also for correlating with health status. Conclusions: Both work stress and family stress should be considered together when addressing the health of working women in the industrial sector in Korea.

Notes: Abstract  Objectives: Inactive nurses' interest in volunteering for emergency preparedness was examined. Methods: A mail survey was sent to the entire Vermont Board of Nursing list of in-state inactive and lapsed registered nurses (n = 3,682). A high rate of undeliverable surveys (60%) was found and 611 surveys were returned for a 20% response rate. Results: Twenty-seven percent of the respondents were interested in volunteering. Those interested in participating in volunteer work as part of a national homeland security effort were significantly more likely to (a) be younger in age (p 〈 0.0001); (b) identify themselves as “being a nurse” (p = 0.001); (c) be employed versus retired (p = 0.002); and (d) be currently volunteers (p = 0.001). Conclusions: Because 33% of the nation's nurses are over age 50, inactive nurses offer a potentially large pool of volunteers for emergency preparedness training and response in the years to come.

Notes: Abstract  Objective : To assess the views of service users towards sexual health nursing outreach clinics situated in youth clubs. The clinics were established in economically deprived areas to meet the needs of young people aged 13–18 yrs who are reluctant to attend mainstream services. Design: Descriptive cross-sectional study. Sample and measurements: 250 questionnaires collecting data on service use and service users’ views of the clinics were distributed to a 25% sample of clinic users. In addition, 20 semi-structured interviews were conducted with service users. Results: 166 questionnaires were returned (66% response rate). The service was equally popular with males and females who often attended to meet friends but subsequently sought sexual health advice. Interviews found that young people appreciated the non-judgmental aspect of the clinics, although some reported concerns about confidentiality when the clinics were busy. Some respondents indicated that the clinics gave them confidence to discuss sexual health matters with others, but often the clinics provided advice for young people who had no one else to talk to. Conclusions: The clinics seem popular with young people, and situating services within an area young people access to socialize appears to be an effective way to target them with sexual health advice.

Notes: Abstract  Optimal home self-management in young children with asthma includes accurate symptom identification followed by timely and appropriate treatment. The objective of this study was to evaluate a home-based asthma educational intervention targeting symptom identification for parents of children with asthma. Two hundred twenty-one children with asthma were enrolled into an ongoing home-based clinical trial and randomized into either a standard asthma education (SAE) or a symptom/nebulizer education intervention (SNEI). Data included home visit records and parent's self-report on questionnaires. Symptom identification and self-management skills significantly improved from preintervention to postintervention for parents in both groups with the exception of checking medications for expiration dates and the frequency of cleaning nebulizer device and equipment. However, significantly more parents of children in the SNEI group reported treating cough symptoms as compared with the SAE group (p = 0.05). Of concern is that only 38% of all parents reported having an asthma action plan in the home. A targeted home-based asthma education intervention can be effective for improving symptom identification and appropriate use of medications in children with asthma. Home asthma educational programs should address accurate symptom identification and a demonstration of asthma medication delivery devices.

Notes: Abstract  The aim of this study was to investigate the relationships between self-reported nocturnal sleep quality and napping patterns in elderly persons with insomnia and to compare the nocturnal sleep quality between napping and non-napping groups. Convenience sampling was used to recruit 60 community-dwelling elderly residents of Taichung City, Taiwan (age range 60–83 years, mean 67.1 years) who reported insomnia. All participants scored greater than 5 on the Pittsburgh Sleep Quality Index (PSQI) questionnaire. Napping prevalence, frequency, and duration were assessed by participant interview. Self-reported sleep quality, sleep latency, sleep duration, sleep efficiency, sleep disturbance, use of sleep medication, and daytime dysfunction were measured with the PSQI. Sixty-four percentage of participants (n = 38) reported napping. There were no age, gender, and ethnicity differences on napping patterns. Global sleep quality, sleep efficiency, and sleep disturbance were significantly associated with prevalence of napping (r = 0.24–0.26, p 〈 0.05). A significant correlation was also found between global sleep quality and nap duration (r = 0.31, p 〈 0.05). Elders in the napping group reported better global sleep quality (t = 2.2, p 〈 0.05) and sleep efficiency (t = 2.1, p 〈 0.05) than those in the non-napping group. The findings suggest that there is no need for health care providers to restrict elderly insomniacs’ daytime napping.

Notes: EDITOR's NOTE  The following reprint of the unsigned editorial for the April 1952 issue of Public Health Nursing describes the historical needs and the continuing development of school health nursing from the early to mid-20th century. Twenty-first century schools continue to deal with some of the same issues such as hunger, poor nutrition, and the adverse effects of overly burdensome work schedules on adolescent health and mental well-being. The goal, so optimistically anticipated by the editors of Public Health Nursing in 1952, of continuous, well-coordinated health supervision from birth to maturity continues to elude us. Of course, school nurses and other health personnel address problems not openly discussed in the 1950s—substance abuse, violence, sexually transmitted diseases, and teen pregnancy. The theme of this historical editorial is the power of advocacy—and the responsibility public health nurses have to use our talents to improve child health.