Heiko Sorg

Tissue Engineered Skin Substitutes Created by Laser-Assisted Bioprinting Form Skin-Like Structures in the Dorsal Skin Fold Chamber in Mice

Tissue engineering plays an important role in the production of skin equivalents for the therapy of chronic and especially burn wounds. Actually, there exists no (cellularized) skin equivalent which might be able to satisfactorily mimic native skin. Here, we utilized a laser-assisted bioprinting (LaBP) technique to create a fully cellularized skin substitute. The unique feature of LaBP is the possibility to position different cell types in an exact three-dimensional (3D) spatial pattern. For the creation of the skin substitutes, we positioned fibroblasts and keratinocytes on top of a stabilizing matrix (Matriderm®). These skin constructs were subsequently tested in vivo, employing the dorsal skin fold chamber in nude mice. The transplants were placed into full-thickness skin wounds and were fully connected to the surrounding tissue when explanted after 11 days. The printed keratinocytes formed a multi-layered epidermis with beginning differentiation and stratum corneum. Proliferation of the keratinocytes was mainly detected in the suprabasal layers. In vitro controls, which were cultivated at the air-liquid-interface, also exhibited proliferative cells, but they were rather located in the whole epidermis. E-cadherin as a hint for adherens junctions and therefore tissue formation could be found in the epidermis in vivo as well as in vitro. In both conditions, the printed fibroblasts partly stayed on top of the underlying Matriderm® where they produced collagen, while part of them migrated into the Matriderm®. In the mice, some blood vessels could be found to grow from the wound bed and the wound edges in direction of the printed cells. In conclusion, we could show the successful 3D printing of a cell construct via LaBP and the subsequent tissue formation in vivo. These findings represent the prerequisite for the creation of a complex tissue like skin, consisting of different cell types in an intricate 3D pattern.

Effects of erythropoietin in skin wound healing are dose related.

The hematopoietic growth factor erythropoietin (EPO) attracts attention due to its all‐tissue‐protective pleiotropic properties. We studied the effect of EPO on dermal regeneration using intravital microscopy in a model of full dermal thickness wounds in the skin‐fold chamber of hairless mice. Animals received repetitive low doses or high doses of EPO (RLD‐EPO or RHD‐EPO) or a single high dose of EPO (SHD‐EPO). SHD‐EPO accelerated wound epithelialization, reduced wound cellularity, and induced maturation of newly formed microvascular networks. In contrast, RHD‐EPO impaired the healing process, as indicated by delayed epithelialization, high wound cellularity, and lack of maturation of microvascular networks. Also, RHD‐EPO caused an excessive erythrocyte mass and rheological malfunction, further deteriorating vessel and tissue maturation. Moreover, RHD‐EPO altered fibroblast and keratinocyte migration in vitro, while both cell types exposed to RLD‐EPO, and, in particular, to SHD‐EPO showed accelerated wound scratch closure. In summary, our data show that a single application of a high dose of EPO accelerates and improves skin wound healing.—Sorg, H., Krueger, C., Schulz, T., Menger, M. D., Schmitz, F., Vollmar, B. Effects of erythropoietin in skin wound healing are dose related. FASEBJ. 23, 3049–3058 (2009). www.fasebj.org

Targeted delivery of human VEGF gene via complexes of magnetic nanoparticle-adenoviral vectors enhanced cardiac regeneration.

This study assessed the concept of whether delivery of magnetic nanobeads (MNBs)/adenoviral vectors (Ad)–encoded hVEGF gene (AdhVEGF) could regenerate ischaemically damaged hearts in a rat acute myocardial infarction model under the control of an external magnetic field. Adenoviral vectors were conjugated to MNBs with the Sulfo-NHS-LC-Biotin linker. In vitro transduction efficacy of MNBs/Ad–encoded luciferase gene (Adluc) was compared with Adluc alone in human umbilical vein endothelial cells (HUVECs) under magnetic field stimulation. In vivo, in a rat acute myocardial infarction (AMI) model, MNBs/AdhVEGF complexes were injected intravenously and an epicardial magnet was employed to attract the circulating MNBs/AdhVEGF complexes. In vitro, compared with Adluc alone, MNBs/Adluc complexes had a 50-fold higher transduction efficiency under the magnetic field. In vivo, epicardial magnet effectively attracted MNBs/AdhVEGF complexes and resulted in strong therapeutic gene expression in the ischemic zone of the infarcted heart. When compared to other MI-treated groups, the MI-M+/AdhVEGF group significantly improved left ventricular function (p<0.05) assessed by pressure-volume loops after 4 weeks. Also the MI-M+/AdhVEGF group exhibited higher capillary and arteriole density and lower collagen deposition than other MI-treated groups (p<0.05). Magnetic targeting enhances transduction efficiency and improves heart function. This novel method to improve gene therapy outcomes in AMI treatment offers the potential into clinical applications.

Skin Wound Healing: An Update on the Current Knowledge and Concepts

Background: The integrity of healthy skin plays a crucial role in maintaining physiological homeostasis of the human body. The skin is the largest organ system of the body. As such, it plays pivotal roles in the protection against mechanical forces and infections, fluid imbalance, and thermal dysregulation. At the same time, it allows for flexibility to enable joint function in some areas of the body and more rigid fixation to hinder shifting of the palm or foot sole. Many instances lead to inadequate wound healing which necessitates medical intervention. Chronic conditions such as diabetes mellitus or peripheral vascular disease can lead to impaired wound healing. Acute trauma such as degloving or large-scale thermal injuries are followed by a loss of skin organ function rendering the organism vulnerable to infections, thermal dysregulation, and fluid loss. Methods: For this update article, we have reviewed the actual literature on skin wound healing purposes focusing on the main phases of wound healing, i.e., inflammation, proliferation, epithelialization, angiogenesis, remodeling, and scarring. Results: The reader will get briefed on new insights and up-to-date concepts in skin wound healing. The macrophage as a key player in the inflammatory phase will be highlighted. During the epithelialization process, we will present the different concepts of how the wound will get closed, e.g., leapfrogging, lamellipodial crawling, shuffling, and the stem cell niche. The neovascularization represents an essential component in wound healing due to its fundamental impact from the very beginning after skin injury until the end of the wound remodeling. Here, the distinct pattern of the neovascularization process and the special new functions of the pericyte will be underscored. At the end, this update will present 3 topics of high interest in skin wound healing issues, dealing with scarring, tissue engineering, and plasma application. Conclusion: Although wound healing mechanisms and specific cell functions in wound repair have been delineated in part, many underlying pathophysiological processes are still unknown. The purpose of the following update on skin wound healing is to focus on the different phases and to brief the reader on the current knowledge and new insights. Skin wound healing is a complex process, which is dependent on many cell types and mediators interacting in a highly sophisticated temporal sequence. Although some interactions during the healing process are crucial, redundancy is high and other cells or mediators can adopt functions or signaling without major complications.

Capsular contracture by silicone breast implants: possible causes, biocompatibility, and prophylactic strategies.

The most common implanted material in the human body consists of silicone. Breast augmentation and breast reconstruction using silicone-based implants are procedures frequently performed by reconstructive and aesthetic surgeons. A main complication of this procedure continues to be the development of capsular contracture (CC), displaying the result of a fibrotic foreign body reaction after the implantation of silicone. For many years, experimental and clinical trials have attempted to analyze the problem of its etiology, treatment, and prophylaxis. Different theories of CC formation are known; however, the reason why different individuals develop CC in days or a month, or only after years, is unknown. Therefore, we hypothesize that CC formation, might primarily be induced by immunological mechanisms along with other reasons. This article attempts to review CC formation, with special attention paid to immunological and inflammatory reasons, as well as actual prophylactic strategies. In this context, the word “biocompatibility” has been frequently used to describe the overall biological innocuousness of silicone in the respective studies, although without clear-cut definitions of this important feature. We have therefore developed a new five-point scale with distinct key points of biocompatibility. Hence, this article might provide the basis for ongoing discussion in this field to reduce single-publication definitions as well as increase the understanding of biocompatibility.

The anti-thrombotic effect of hydrogen sulfide is partly mediated by an upregulation of nitric oxide synthases.

INTRODUCTION Hydrogen sulfide (H2S) known as a gasotransmitter is increasingly recognized for its anti-adhesive, anti-inflammatory and vasoactive properties. Due to these properties, we analysed anti-thrombotic effects of H2S and the participation of the nitric oxide synthase (NOS)-pathway. MATERIALS AND METHODS In individual venules of the ear of hairless SKH1-hr mice, thrombus formation was induced using a phototoxic light/dye-injury model and intravital fluorescence microscopy. Animals were treated intravenously with the H2S donor Na2S or NaCl as control. In a second setting, the NOS inhibitor L-NAME was applied intraperitoneally as a bolus 12h prior to Na2S treatment and thrombus induction. Blood and ear tissue were sampled after microscopy for assessment of plasma concentrations of soluble (s)P-selectin, sE-selectin, sVCAM-1 and sICAM-1 and expression of endothelial (e)NOS and inducible (i)NOS, respectively. RESULTS When mice were treated with Na2S, venular thrombus formation was significantly delayed versus that in animals of the NaCl-treated control group. While plasma levels of pro-thrombotic adhesion molecules were not affected by Na2S, immunohistochemistry of the vessel walls showed a significant up-regulation of eNOS and iNOS expression within the Na2S-treated group. The delay of thrombus formation in the Na2S-group was partly but significantly reverted by application of L-NAME. CONCLUSIONS The anti-thrombotic efficacy of H2S involves the NOS-pathway and may be of preventive and therapeutic value for clinical disorders with increased risk of thrombotic events.

The nonhematopoietic effects of erythropoietin in skin regeneration and repair: from basic research to clinical use.

Erythropoietin (EPO) is the main regulator of red blood cell production but there exists also a variety of nonhematopoietic properties. More recent data show that EPO is also associated with the protection of tissues suffering from ischemia and reperfusion injury as well as with improved regeneration in various organ systems, in particular the skin. This review highlights the mechanisms of EPO in the different stages of wound healing and the reparative processes in the skin emphasizing pathophysiological mechanisms and potential clinical applications. There is clear evidence that EPO effectively influences all wound‐healing phases in a dose‐dependent manner. This includes inflammation, tissue, and blood vessel formation as well as the remodeling of the wound. The molecular mechanism is predominantly based on an increased expression of the endothelial and inducible nitric oxide (NO) synthase with a consecutive rapid supply of NO as well as an increased content of vascular endothelial growth factor (VEGF) in the wound. The improved understanding of the functions and regulatory mechanisms of EPO in the context of wound‐healing problems and ischemia/reperfusion injury, especially during flap surgery, may lead to new considerations of this growth hormone for its regular clinical application in patients.

The mouse dorsal skin fold chamber as a means for the analysis of tissue engineered skin

The therapy of extensive and deep burn wounds is still a challenging task for reconstructive plastic surgery. The outcome is generally not satisfactory, neither from the functional nor from the aesthetic aspect. Several available skin substitutes are used but there is need for optimization of new skin substitutes which have to be tested in vitro as well as in vivo. Here, we show that the dorsal skin fold chamber preparation of mice is well suited for the testing of skin substitutes in vivo. Dermal skin constructs consisting of matriderm(®) covered with a collagen type I gel were inserted into full thickness skin wounds in the skin fold chambers. The skin substitutes integrated well into the adjacent skin and got epithelialized from the wound edges within 11 days. The epithelialization by keratinocytes is the prerequisite that also cell-free dermal substitutes might be used in the case of the lack of sufficient areas to gain split thickness skin grafts. Further advantage of the chambers is the lack of wound contraction, which is common but undesired in rodent wound healing. Furthermore, this model allows a sophisticated histological as well as immunohistochemical analysis. As such, we conclude that this model is well suited for the analysis of tissue engineered skin constructs. Besides epithelialization the mode and extend of neovascularization and contraction of artificial grafts may be studied under standardized conditions.

Early rise in inflammation and microcirculatory disorder determine the development of autoimmune pancreatitis in the MRL/Mp-mouse

Autoimmune pancreatitis (AIP) is a rare cause of chronic pancreatitis and mimics pancreatic cancer. Although there is strong interest in research, etiology and pathophysiology of AIP are still unknown. Therefore, we analyzed a total of 92 MRL/Mp-mice of either sex, which are prone to develop AIP, in four different age groups (8-12, 16-20, 24-28, and 32-40 wk). Using intravital fluorescence microscopy, histology, laboratory analysis, and Western blot, onset, severity, and pathophysiological mechanisms of AIP were evaluated. Female animals showed in vivo an age-dependent increase of intrapancreatic leukocyte accumulation, as well as a loss in functional capillary perfusion. In contrast, intrapancreatic inflammation in male mice was less pronounced and not age dependent. Furthermore, pancreatic tissue specimen of female animals exhibited major organ destruction with significantly higher values of mean pathological scores (1.5 +/- 0.3 vs. < or =0.2; P < 0.05), as well as significantly increased CD4-, CD8-, CD11b-, and CD138-positive cells compared with male animals of the same age. Interestingly, there was a significant positive correlation between intravascular leukocyte adherence and the histopathological score of the pancreas, indicating a determining role of the innate immune system for the late onset of AIP. The present study shows that the onset of AIP is characterized by an inflammatory response and microcirculatory failure, most probably constituting initiators and propagators of this autoimmune disease.

Quantitative evaluation of the requirements for the promotion as associate professor at German medical faculties.

Background: First quantitative evaluation of the requirements for the promotion as associate professor (AP) at German Medical Faculties Material and methods: Analysis of the AP-regulations of German Medical Faculties according to a validated scoring system, which has been adapted to this study. Results: The overall scoring for the AP-requirements at 35 German Medical Faculties was 13.5±0.6 of 20 possible scoring points (95% confidence interval 12.2-14.7). More than 88% of the AP-regulations demand sufficient performance in teaching and research with adequate scientific publication. Furthermore, 83% of the faculties expect an expert review of the candidate´s performance. Conference presentations required as an assistant professor as well as the reduction of the minimum time as an assistant professor do only play minor roles. Conclusion: The requirements for assistant professors to get nominated as an associate professor at German Medical Faculties are high with an only small range. In detail, however, it can be seen that there still exists large heterogeneity, which hinders equal opportunities and career possibilities. These data might be used for the ongoing objective discussion.