Bjoern Menger

Interactions between Spider Silk and Cells – NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames

Background Several materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature. Methodology/Principal Findings Steel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen- and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01). Conclusion/Significance Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres.

Resection of a Large Intra-Abdominal Tumor in the Mexican Axolotl: A Case Report

OBJECTIVE This case report describes the surgical removal of an intra-abdominal tumor from a Mexican axolotl (Ambystoma mexicanum). The animal was admitted with left abdominal swelling that had increased over 4 months. METHODS Surgical removal was performed under general anesthesia with MS222 under an operating microscope. Exploratory laparotomy was performed through 2.5 dorsocranial skin incision in the left flank, followed by subcutaneous dissection. RESULTS The tumor involved the spleen, was adjacent to the descending colon, and supplied by vessels from the spleen, stomach, and colon. The mass was removed by clamping and transecting the spleen and the peritoneum was closed with a continuous suture pattern, while abdominal muscles and skin were closed in layers. After a total duration time of anesthesia of 90 minutes the animal was kept in prophylactic antibiotic baths. Tissue sections revealed characteristics of both lymphangiosarcoma and lymphosarcoma with an appearance typical for a malignant tumor. CONCLUSIONS Abdominal surgery was performed in an axolotl and the surgical wound healed without complication.

Applying amphibian limb regeneration to human wound healing: a review.

In contrast to the limited regenerative ability found in human wound healing, which often results in unsatisfying and deficient scar formation, urodele amphibians, with the Mexican axolotl as a prime example, expose an extraordinary regenerative capacity. This regeneration leads to a perfect restoration of tissue architecture, function, and aesthetics with the axolotl being actually able to reclaim complete limbs. Evolutionary considerations suggest that regeneration might be a biologic principle which also underlies human wound healing. Experimental findings, such as comparative studies on transforming growth factor-&bgr; and fibroblast growth factor accentuate this assumption. Regeneration, as recent data indicate, might be a question of adaptive immunity. The loss of regenerative potency correlates with the decrease of regeneration in most species, whereas the Mexican axolotl lacks adaptive immunity throughout its life. The characterization of molecular pathways as a prerequisite for any control of regenerative processes sets an increasing indication toward the transfer into human beings. Some regenerative techniques, eg, recombinant transforming growth factor-&bgr; have already emerged. Molecular findings suggest that there is an intrinsic regenerative capacity in humans which might be initiated under appropriate circumstances. The Mexican axolotl is liable to diverse surgical and molecular approaches. Though well-known among developmental biologists, its exploitation for experimental Plastic Surgery still has to be established. We therefore intend to give an introduction to amphibian regeneration and the common evolutionary roots of regeneration and human wound healing, as we believe that Plastic Surgery takes a unique advantage of performing basic research on amphibian regeneration.

AmbLOXe--an epidermal lipoxygenase of the Mexican axolotl in the context of amphibian regeneration and its impact on human wound closure in vitro.

OBJECTIVE The Mexican axolotl (Ambystoma mexicanum) is a well-characterized example for intrinsic regeneration. As lipoxygenase signaling is of crucial importance to scarless mammalian wound healing, we postulated that lipoxygenases might be expressed during amphibian regeneration and they might also influence human cells under appropriate conditions. In this study we identified an amphibian lipoxygenase and evaluated its impact on human cells in an in vitro wound model. METHODS cDNA encoding for amphibian epidermal lipoxygenase (AmbLOXe) was polymerase chain reaction amplified and sequenced followed by phylogenic classification based on T-coffee alignment. Distribution of AmbLOXe was examined in various Ambystoma tissues, using polymerase chain reaction and in situ hybridization. Lipoxgenase influence was investigated using an outgrowth model of amphibian epidermal cells. Human osteosarcoma, as well as keratinocyte cell lines expressing AmbLOXe, were tested concerning in vitro wound closure in a monolayer scratch model. RESULTS We isolated AmbLOXe from Ambystoma limb bud blastema identified as a homologue of human epidermal lipoxygenase. Amphibian epidermal lipoxygenase is expressed in Axolotl limb blastema and in epidermal cells which show decreased cell migration and proliferation rates when treated with LOX inhibitors. Furthermore, human osteosarcoma and keratinocyte cells showed increased rates of cell migration if transfected with AmbLOXe. CONCLUSION In this study, AmbLOXe, a new effector of amphibian regeneration is described. In consideration of the presented data, AmbLOXe is important for amphibian epidermal cell proliferation and migration. As AmbLOXe expressing human osteosarcoma and keratinocyte cell lines showed increased rates of in vitro wound closure, an influence of amphibian mediators on human cells could be described for the first time.

A novel and inexpensive retractor individually usable for microsurgical training.

Inspired by a study published in this journal some years ago by Narayanan and Ramasastry, this letter is to introduce a new kind of retractors utilizable in microsurgical training. Although a recent publication seized the concept of designing inexpensive microretractors using common supplies, we found certain disadvantages using both of the microretractors mentioned beforehand. Mainly, these disadvantages are lack of stiffness and oxidation caused by the sterilizing process, our aim was to overcome these problems by using medical products of certain stiffness. Spring hard dental straight wires (0.7-mm thick) were purchased inexpensively from Dentaurum in a length of 30 cm (REF 527-070-00, Dentaurum, Ispringen, Germany). A three-point wire bending plier purchased from the same manufacturer was utilized to bend wires to the desired shape (REF 003-200-00, Dentaurum, Ispringen, Germany). Different shapes were bent to obtain the ideal retractor for different tissues. The unmodified end of the retractor was bent in a spiral manner, thus it could be clipped to a simple metal frame which was laid on the operation platform (Fig. 1A). As no greater forces have to be dealt with in microsurgical training, we found our retractors to be sufficiently secured for all microsurgical applications by the weight of the frame. In this manner the hook was applicable in every region of the operation area, including deep microsurgery (Fig. 1B). Additionally, a surgical clamp could be fixed as weight to the distal end of the retractor or, if possible, it could simply be pierced in the operation platform. We could never observe any damage in the retracted tissue, which we attributed to the soft edges of the hook-like ends of the retractors. As the wire leaves the operation field in a divergent manner while the bent angle is close to the superficial edges of the operation situs, the entrance to the operation field is as opened and wide as possible (Fig. 1A). No hindering of entrance of instruments and no rotating or twisting of the wire could be observed, probably because of the spring-hard stiffness. Comparing our approach to the use of staples or paper clips in microsurgical training, it has to be noted that staples display a very short length and thus may be affected by restrictions. While these could be secured just by their own weight and by the use of sterile clamps, our retractors granted superior security according to the different and more sufficient possibilities of fixation. Due to their relative moldability, they can easily be deformed accidentally. In contrast, the microretractors presented here were spring-hard and thus accidental deformation could be avoided. Additionally, they are medical products which are not oxidated even after more than one sterilizing processes, as staple or paper clip retractors may do. Spring-tightened microretractor, as previously described, were especially designed for microvascular surgery with hooks on both ends. This special design also restricts their usage to other fields of microsurgery. In contrast, the microretractors developed here could be used in a more universal manner because of their lengths and shapes of the retracting ends, which could be adapted easily to multiple settings (Fig. 1B). The adjustable microretractors presented in this letter might be helpful in microsurgical training by optimizing the exposition of the *Correspondence to: Jörn W. Kuhbier, Department for Plastic, Hand und Reconstructive Surgery, Laboratory for Experimental Plastic Surgery, Hannover Medical School, Podbielskistraße 380, 30659 Hannover. E-mail: joernkuhbier@gmx.de Received 18 April 2010; Accepted 5 October 2010 Published online 28 January 2011 in Wiley Online Library (wileyonlinelibrary. com). DOI 10.1002/micr.20853

The yeast two hybrid system in a screen for proteins interacting with axolotl (Ambystoma mexicanum) Msx1 during early limb regeneration.

Urodele amphibians are exceptional in their ability to regenerate complex body structures such as limbs. Limb regeneration depends on a process called dedifferentiation. Under an inductive wound epidermis terminally differentiated cells transform to pluripotent progenitor cells that coordinately proliferate and eventually redifferentiate to form the new appendage. Recent studies have developed molecular models integrating a set of genes that might have important functions in the control of regenerative cellular plasticity. Among them is Msx1, which induced dedifferentiation in mammalian myotubes in vitro. Herein, we screened for interaction partners of axolotl Msx1 using a yeast two hybrid system. A two hybrid cDNA library of 5-day-old wound epidermis and underlying tissue containing more than 2×10⁶ cDNAs was constructed and used in the screen. 34 resulting cDNA clones were isolated and sequenced. We then compared sequences of the isolated clones to annotated EST contigs of the Salamander EST database (BLASTn) to identify presumptive orthologs. We subsequently searched all no-hit clone sequences against non redundant NCBI sequence databases using BLASTx. It is the first time, that the yeast two hybrid system was adapted to the axolotl animal model and successfully used in a screen for proteins interacting with Msx1 in the context of amphibian limb regeneration.

LP34: AMPHIBIAN EPIDERMAL LIPOXYGENASE AMBLOXE ENHANCES MAMMALIAN WOUND HEALING IN VIVO

The Mexican axolotl is capable to regenerate even whole limbs in succession of amputation. In our recent works we cloned and characterized an amphibian epidermal lipoxygenase (AmbLOXe) from axolotl regenerating tissue and showed its influence on human wound healing in vitro (1). In this work we intend to evaluate its effect on mammalian wound closure in vivo.

Preliminary investigations of spider silk in wounds in vivo — Implications for an innovative wound dressing

The ideal wound dressing in particular for burn wounds has not been found yet. The aim of this study was to investigate native spider silk as a novel wound dressing. Release of inflammatory cytokines of macrophages and neutrophile granulocytes was determined via ELISA after exposure to spider silk. Migration of dermal cells as well as angiogenesis on spider silk was visualized with live video microscopy or chorioallantois membrane model, respectively. Native spider silk was placed in full-thickness skin wounds in a sheep in vivo-model and wounds were evaluated after 2, 4, 6, and 8weeks histologically as well as per quantitative real-time PCR. Minimal inflammatory cytokine release could be seen for spider silk. Ingrowth of single capillaries into bundles of spider silk and migration of keratinocytes as well as fibroblasts on spider silk fibres was proven. Macroscopically, a comparable wound closure could be seen in spider silk and in sham controls. In histological evaluation, a thicker epidermis was observed in spider silk treated wounds while collagen III/I expression ratio was comparable in both groups. As native spider silk has been described as highly biocompatible, it might represent an innovative alternative to common wound dressings.

162A: OUTGROWTH OF NEW FINGERS IN FLANKS OF THE MEXICAN AXOLOTL INDUCED BY BONE AUTOTRANSPLANTATION - A PROMISING FUTURE MODEL FOR PLASTIC AND RECONSTRUCTIVE SURGERY

Introduction: In contradiction to amphibians, humans display limited regenerative capacity leading to serious restrictions in Reconstructive Surgery. Adult urodeles are able to regrow many lost structures; it has been reported that limb regeneration depends on an inductive wound epithelium and appropriate nerve supply. All studies published up to date characterize the participation of cell and tissue components to this process by experimental interferences at the amputation site or by artificially creating situations alike. In this study, we intended to investigate the regenerative capacity of amphibian bone tissue in a standardized environment without adaption to the amputational setting.