Giorgio Pietramaggiori

The mechanism of action of the vacuum-assisted closure device.

Background: The vacuum-assisted closure device is widely used clinically, yet its mechanisms of action are incompletely understood. In this study, the authors designed a partially splinted full-thickness murine vacuum-assisted closure model to better understand the mechanism of action of the vacuum-assisted closure device. Methods: Full-thickness wounds (n = 10 per group) were excised in diabetic mice and treated with the vacuum-assisted closure device or its isolated components: an occlusive dressing, subatmospheric pressure at 125 mmHg (suction), and a polyurethane foam without and with downward compression. Results were quantified with a two-dimensional immunohistochemical staging system based on blood vessel density (CD31) and cell proliferation (Ki67) 7 days after wounding. Microscopic strain was measured by fixing in situ all dressing modalities. Results: Wounds exposed to polyurethane foam in compressed and uncompressed dressings or to the vacuum-assisted closure device showed a 2-fold increase in vascularity compared with the occlusive dressing group (p < 0.05). The vacuum-assisted closure device in addition stimulated cell proliferation, with up to 82 percent Ki67-positive nuclei, compared with the other groups. Direct measurements of wound surface deformations showed significant microstrains in the vacuum-assisted closure and foam in compressed dressing groups (60 percent and 16 percent, respectively) compared with all other groups. Conclusions: These data provide profound insights into the mechanism of action of the vacuum-assisted closure device, providing an explanation for the increases in wound bed vascularity and cell proliferation based on its components. Results suggest that the vascular response is related to the polyurethane foam, whereas tissue strains induced by the vacuum-assisted closure device stimulated cell proliferation.

The Transcription Factor EGR1 Controls Both the Proliferation and Localization of Hematopoietic Stem Cells

EGR1 is a member of the immediate early response transcription factor family and functions in cell growth, development, and stress responses in many tissues. Here we report an additional role for EGR1 in regulating homeostasis of hematopoietic stem cells (HSCs). HSCs normally express Egr1 at high levels, but dramatically downregulate its expression when induced to divide and migrate. Consistent with this finding, mice lacking Egr1 exhibit significant increases in steady-state levels of dividing HSCs in the bone marrow (BM), and a striking spontaneous mobilization of HSCs into the peripheral blood. These data identify EGR1 as a transcriptional regulator of stem cell migration that normally functions to promote HSC quiescence and retention in the niche. The ability of this single factor to regulate both proliferation and mobilization of HSCs suggests that EGR1 commands a genetic program that coordinates stem cell division and migration to maintain appropriate HSC number and function.

Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

Tensile forces stimulate vascular remodeling and epidermal cell proliferation in living skin.

Objectives:To quantify tissue remodeling induced by static and cyclical application of tensional forces in a living perfused tissue. Background:Cells are able to respond to mechanical cues from the environment and can switch between proliferation and quiescence. However, the effects of different regimens of tension on living, perfused skin have not been characterized. Methods:The ears of living rats were mechanically loaded by applying tensile forces (0.5 Newtons) either statically or cyclically and then analyzing tissue responses using in vivo microscopy, immunohistochemistry, and corrosion casting. Results:Quantitative immunohistochemistry showed that in the static group (4-day continuous tension) there was up to 4-fold increase in cellular proliferation in the epidermis after 4 days and a 2.8-fold increase in the vascularity in the dermis that peaked after 2 days. Comparable effects could be achieved in just 8 hours using a cyclic loading protocol. We also modeled the resultant stress produced in the ear using a linear finite element model and demonstrated a correlation between the level of applied stress and both epidermal cell proliferation and blood vessel density. Conclusions:Mechanical forces stimulate cell proliferation and vascular remodeling in living skin. As cell growth and vascular supply are critical to wound healing and tissue expansion, devices applying controlled mechanical loads to tissues may be a powerful therapy to treat tissue defects.

Freeze-dried platelet-rich plasma shows beneficial healing properties in chronic wounds.

Fresh platelet concentrates are used in many centers to treat recalcitrant wounds. To extend the therapeutic shelf‐life of platelets, we analyzed the wound‐healing effects of fresh‐frozen and freeze‐dried (FD) platelet‐rich plasma (PRP) using a diabetic mouse model. Db/db mice with 1.0 cm2 dorsal excisional wounds (n=15/group) were treated with a single application of FD PRP (1.2 × 106 platelets/μL) with or without a stabilization solution, and compared with wounds treated with fresh‐frozen, sonicated PRP, and untreated wounds. Granulation tissue area, thickness, and wound size were analyzed 9 days posttreatment. Immunostained sections were quantified for vascularity and proliferation using antiplatelet endothelial cell adhesion molecule I and antiproliferating cell nuclear antigen antibodies. The results showed that all PRP preparations increased granulation tissue formation as assessed by surface coverage, thickness, and angiogenic response, when compared with untreated wounds. In addition, wounds treated with FD PRP, and biochemically stabilized FD PRP, exhibited higher proliferative levels. The possibility to deliver growth factors using platelets, and the potential to extend the shelf‐life of platelet concentrates makes freeze‐drying methods particularly suitable for enhanced wound care.

A review of the role of mechanical forces in cutaneous wound healing.

Cutaneous wound healing is a complex process with many types of mechanical forces regulating the quality and speed of healing. The role of mechanical forces in regulating tissue growth, repair and remodelling was recognised more than a century ago. Such forces influence gene expression, the synthesis of growth factors and inflammatory mediators and cellular processes like proliferation of many load-sensitive cells. However, the exact mechanisms by which these forces interact with cells and ways to use them to stimulate tissues are still active research fronts. This article sets to review the literature on mechanical forces and their role in cutaneous wound healing.

Healing modulation induced by freeze-dried platelet-rich plasma and micronized allogenic dermis in a diabetic wound model

The incidence and prevalence of chronic and diabetic wounds are increasing and clinical treatments to tackle these epidemics are still insufficient. In this study, we tested the ability of freeze‐dried platelet‐rich plasma (PRP) and an allogenic micronized acellular dermal matrix alone and in combination to modulate diabetic wound healing. Therapeutic materials were applied to 1.0 cm2 excisional wounds on genetically diabetic (db/db) mice. Wound‐healing kinetics and new tissue formation were studied at 9 and 21 days posttreatment. Quantitative immunohistochemistry was used to study vascularity and cellular proliferation (days 9 and 21), and collagen deposition was evaluated 21 days postwounding. In vitro, micronized allogenic dermis, when combined with PRP, absorbed nearly 50% of original platelet‐derived growth factor, transforming growth factor‐β, vascular endothelial growth factor, and epidermal growth factor from platelets and stimulated fibroblast proliferation. In vivo, micronized dermis increased the formation of vascularized wound tissue by day 9. Freeze‐dried PRP alone or in combination with micronized dermis increased wound tissue revascularization and proliferation compared with spontaneous healing. The increase in cell proliferation persisted until day 21 only when freeze‐dried PRP was used in combination with micronized dermis. These results indicate that micronized allogenic dermis may be used to provide a dermal matrix to stimulate tissue formation and the combination with PRP may confer additional beneficial growth factors to chronic or diabetic wounds.

Effect of Recombinant Platelet-Derived Growth Factor (Regranex®) on Wound Closure in Genetically Diabetic Mice

Burns, especially those involving large surface areas, represent a complex wound healing problem. Platelet-derived growth factor (PDGF) is released by activated platelets to recruit inflammatory cells toward the wound bed. It has effects on promoting angiogenesis and granulation tissue formation. However, the effectiveness of topical PDGF on wound closure is variable, ranging from little improvement observed in pig models to dramatic improvement reported in a diabetic mouse model. Here, we sought to determine the effectiveness of commercially sold PDGF-BB (Regranex®) on wound closure in genetically diabetic mice. C57BL/KsJ db+/db+ mice and its host strain bearing dorsal 1.5-cm2 wounds were divided into groups (n = 8 in each group) receiving topical application of either Regranex® (10 &mgr;g/wound) or vehicle for 5 consecutive days after wounding. The rate of wound closure was analyzed using computerized planimetry. The amount of granulation tissue was determined histologically. Our data indicate that diabetic mice exhibit a significant delay in wound closure when compared with their host strain. Topical application of Regranex® did not improve the time to wound closure but did significantly increase the amount of granulation tissue. Our current study using commercially available Regranex® failed to reproduce the previously reported finding that PDGF improved wound closure in healing impaired genetically diabetic mice.

Hypoxia Impairs Skin Myofibroblast Differentiation and Function

Ischemic wounds are characterized by oxygen levels lower than that of healthy skin (hypoxia) and poor healing. To better understand the pathophysiology of impaired wound healing, we investigated how switching from high (21%) to low (2%) oxygen levels directly affects cultured skin myofibroblasts, essential cells for the normal wound repair process. Myofibroblast differentiation and function were assessed by quantifying α-smooth muscle actin expression and cell contraction in collagen gels and on wrinkling silicone substrates. Culture for 5 days at 2% oxygen is perceived as hypoxia and significantly reduced myofibroblast differentiation and contraction despite high levels of the profibrotic transforming growth factor-β1. Analysis of α-smooth muscle actin expression on wrinkling substrates over time showed that reduced myofibroblast contraction preceded α-smooth muscle actin disassembly from stress fibers after switching from 21 to 2% oxygen. These effects were reversible by restoring high oxygen conditions and by applying mechanical stress. We suggest that mechanical challenge is a clinical relevant strategy to improve ischemic and chronic wound healing by supporting myofibroblast formation.

Poly-N-Acetyl Glucosamine Nanofibers: A New Bioactive Material to Enhance Diabetic Wound Healing by Cell Migration and Angiogenesis

Introduction:In several fields of surgery, the treatment of complicated tissue defects is an unsolved clinical problem. In particular, the use of tissue scaffolds has been limited by poor revascularization and integration. In this study, we developed a polymer, poly-N-acetyl-glucosamine (sNAG), with bioactive properties that may be useful to overcome these limitations. Objective:To develop a scaffold-like membrane with bioactive properties and test the biologic effects in vitro and in vivo in diabetic wound healing. Methods:In vitro, cells–nanofibers interactions were tested by cell metabolism and migration assays. In vivo, full thickness wounds in diabetic mice (n = 15 per group) were treated either with sNAG scaffolds, with a cellulosic control material, or were left untreated. Wound healing kinetics, including wound reepithelialization and wound contraction as well as microscopic metrics such as tissue growth, cell proliferation (Ki67), angiogenesis (PECAM-1), cell migration (MAP-Kinase), and keratinocyte migration (p 63) were monitored over a period of 28 days. Messenger RNA levels related to migration (uPAR), angiogenesis (VEGF), inflammatory response (IL-1β), and extracellular matrix remodeling (MMP3 and 9) were measured in wound tissues. Results:sNAG fibers stimulated cell metabolism and the in vitro migratory activity of endothelial cells and fibroblasts. sNAG membranes profoundly accelerated wound closure mainly by reepithelialization and increased keratinocyte migration (7.5-fold), granulation tissue formation (2.8-fold), cell proliferation (4-fold), and vascularization (2.7-fold) compared with control wounds. Expression of markers of angiogenesis (VEGF), cell migration (uPAR) and ECM remodeling (MMP3, MMP9) were up-regulated in sNAG treated wounds compared with controls. Conclusions:The key mechanism of the bioactive membranes is the cell-nanofiber stimulatory interaction. Engineering of bioactive materials may represent the clinical solution for a number of complex tissue defects.